Cooperative primers, probes, and applications thereof

ABSTRACT

Disclosed are compositions and a method relating to amplifying and detecting nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/944,192, filed Jul. 17, 2013, which claims benefit of U.S.Provisional Application No. 61/672,329, filed Jul. 17, 2012, and61/732,537, filed Dec. 3, 2012, each of which are hereby incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The disclosed invention is generally in the field of nucleic acidamplification in its entirety.

BACKGROUND OF THE INVENTION

Nucleic acid testing often requires amplification of nucleic acids toachieve a sufficient concentration and/or purity to undergo subsequenttesting. Sometimes amplification of nucleic acids is used as a surrogatein detection of non-nucleic acids, such as proteins. The majority ofnucleic acid amplification/extension reactions depend on the presence ofa primer comprised of modified or natural nucleic acids at the 3′ endwhich allow extension in the presence of a polymerase.

A universal problem with such amplification reactions is the presence ofprimer-dimers. Primer-dimers are formed when primers extend each otherrather than the target nucleic acid. Primer-dimers use up primers,resulting in the presence of impurities in the reaction. Even worse,primer-dimers can use up enough primers to cause false negatives in somecases. Or, if interacting with a probe, primer-dimers can cause falsepositives.

A variety of hot starts have been developed to deal with the issue ofprimer-dimers including suspending the polymerase in a wax material,inhibiting the polymerase with antibodies, chemically modifying thepolymerase, sequestering primers, and a variety of other methods. Theproblem with all of these methods is that they are only effective priorto the first round of amplification/extension. Any primer-dimers thatform thereafter are amplified at an exponential rate.

Other methods of dealing with primer-dimers include methods such asnested PCR. However, this requires two separate reactions and increasesthe chances of contamination.

Many amplification/extension reactions are also coupled with a detectionprobe. The principles often revolve around a labeled linear probe, suchas Taqman or a labeled hairpin probe such as Molecular Beacons. Somemethods achieve incredible specificity through the use of cooperativelylinking two probes, such as Tentacle Probes. However, each of theseprobe based methods is limited in detecting mutants in a high backgroundof wild type. While they can achieve all or nothing detection of singlenucleotide polymorphisms and other mutations, they can only pick outabout one mutant in a background of 10 to 20 wild type sequences. Thisis because the primers amplify both the wild type and the mutant and aredepleted without being able to detect both. Methods like ARMS can becombined with the probe detection technologies to overcome this problemto an extent, but cannot be effectively multiplexed for real-timedetection when more than one mutation occurs in the same general region.

Several primers have been developed which include a detection mechanism,such as Amplifluor primers, Rapid Detex primers and Scorpion primers.The first two are especially prone to false positives from primer-dimerproblems because they are not sequence specific. The latter is aself-probing primer, where the probe binds to the primer extensionproduct rather than the nucleic acid template. Because it has a sequencespecific probe, it is less likely to result in false positives, but isstill subject to primer-dimer associated problems.

BRIEF SUMMARY OF THE INVENTION

Disclosed herein is a cooperative nucleic acid molecule comprising: a) afirst nucleic acid sequence, wherein the first nucleic acid sequence issubstantially complementary to a first region of a target nucleic acid,and wherein the first nucleic acid sequence is extendable on the 3′ end;b) a second nucleic acid sequence, wherein the second nucleic acidsequence is substantially complementary to a second region of the targetnucleic acid; wherein the first and second nucleic acid sequences areattached to each other; and wherein the second nucleic acid sequencehybridizes to the target nucleic acid sequence downstream from the 3′end of the first nucleic acid sequence.

Further disclosed is a method for amplifying a target nucleic acid, themethod comprising: a) providing a cooperative nucleic acid molecule asdisclosed herein; b) providing a target nucleic acid; and c) amplifyingthe target nucleic acid under appropriate conditions for amplification;thereby amplifying the target nucleic acid.

Also disclosed is a method for detecting a nucleic acid in a sample, themethod comprising a) providing a cooperative nucleic acid molecule asdisclosed herein, wherein the cooperative nucleic acid comprises adetectable label; b) providing a target nucleic acid; and c) detectingthe target nucleic acid; thereby detecting the target nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of cooperative primers that has a linkerinternal to the primer attached to the 5′ end of the capture sequence.The capture sequence binds to the target nucleic acid, and thehybridized capture sequence holds the primer in close proximity to thetarget. The primer then extends, cleaving the capture sequence.

FIG. 2 shows another embodiment of cooperative primers that has a linkerattaching the 5′ end of the primer to the 3′ end of the capturesequence. The capture sequence binds to the target nucleic acid. Thehybridized capture sequence holds the primer in close proximity to thetarget. The primer then extends, cleaving the capture sequence.

FIG. 3 shows a preferred embodiment of a cooperative primer with the 5′end of the capture sequence linked to the 5′ end of the primer. Thecapture sequence binds to the target nucleic acid. The hybridizedcapture sequence holds the primer in close proximity to the target. Theprimer then extends, cleaving the capture sequence.

FIG. 4 shows examples of probes that can be linked to the primerinclude, but are not limited to, dual labeled probes, hairpin probes andsingle label probes.

FIG. 5 shows a preferred embodiment for detection of nucleic acidextension using a cooperative primer linked to a dual labeled probe. Theprobe binds to the target nucleic acid, and the hybridized probe holdsthe primer in close proximity to the target. The primer extends,cleaving the probe, causing an increase in fluorescence.

FIG. 6A-B shows gel of Cooperative Primers and Normal Primers. FIG. 6Ashows normal primers have some primer-dimer (P-D) formation even when noP-D are spiked in, however, still 25 have amplification of 60 startingcopies of Malaria DNA. When 600 P-D are spiked in, Malaria amplificationproducts are eclipsed and only P-D are amplified. In contrast, FIG. 6Bshows Cooperative Primers have no primer-dimer amplification, even whenup to 600,000 P-D are spiked in. P-D do not interfere with cooperativeprimer amplification of the target nucleic acid.

FIG. 7 shows some examples of primers with built in detection mechanismsthat can be used in cooperative primers.

FIG. 8 shows cooperative primers with integrated probes. Labeledcooperative primers or normal hybridization probes were used for realtime detection of 5,000,000, 50,000, 500 or 0 copies P. falciparumtemplate. Labeled capture sequences in cooperative primers had afluorescent signal 2.5× higher than normal hybridization probes, eventhough the capture sequence had a Tm below the reaction temperature.

FIGS. 9A-B show SNP differentiation with cooperative primers.Cooperative Primers differentiate between Tuberculosis Complex with therpoB D516V SNP causing rifampicin resistance and without the SNP usingprobe based differentiation with the SNP under the capture sequence (9A)and the ARMS based method with the SNP under the 3′ end of the primer(9B).

DETAILED DESCRIPTION OF THE INVENTION

The disclosed method makes use of certain materials and procedures whichallow amplification of nucleic acid sequences and whole genomes or otherhighly complex nucleic acid samples. These materials and procedures aredescribed in detail below.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. The following definitionssupplement those in the art and are directed to the current applicationand are not to be imputed to any related or unrelated case, e.g., to anycommonly owned patent or application. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice for testing of the present invention, the preferred materialsand methods are described herein. Accordingly, the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting.

As used herein, “nucleic acid sequence” refers to the order or sequenceof nucleotides along a strand of nucleic acids. In some cases, the orderof these nucleotides may determine the order of the amino acids along acorresponding polypeptide chain. The nucleic acid sequence thus codesfor the amino acid sequence. The nucleic acid sequence may besingle-stranded or double-stranded, as specified, or contain portions ofboth double-stranded and single-stranded sequences. The nucleic acidsequence may be composed of DNA, both genomic and cDNA, RNA, or ahybrid, where the sequence comprises any combination of deoxyribo- andribo-nucleotides, and any combination of bases, including uracil (U),adenine (A), thymine (T), cytosine (C), guanine (G), inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. It may include modifiedbases, including locked nucleic acids, peptide nucleic acids and othersknown to those skilled in the art.

An “oligonucleotide” is a polymer comprising two or more nucleotides.The polymer can additionally comprise non-nucleotide elements such aslabels, quenchers, blocking groups, or the like. The nucleotides of theoligonucleotide can be natural or non-natural and can be unsubstituted,unmodified, substituted or modified. The nucleotides can be linked byphosphodiester bonds, or by phosphorothioate linkages, methylphosphonatelinkages, boranophosphate linkages, or the like.

A “peptide nucleic acid” (PNA) is a polymer comprising two or morepeptide nucleic acid monomers. The polymer can additionally compriseelements such as labels, quenchers, blocking groups, or the like. Themonomers of the PNA can be unsubstituted, unmodified, substituted ormodified.

By “cooperative nucleic acid” is meant a nucleic acid sequence whichincorporates minimally a first nucleic acid sequence and a secondnucleic acid sequence, wherein the second nucleic acid sequencehybridizes to the target nucleic acid downstream of the 3′ end of thefirst nucleic acid sequence. The 3′ end of the nucleic acid can beextendable, as discussed elsewhere herein. In one example, the firstnucleic acid is a primer, and the second nucleic acid is a capturesequence. The first and second nucleic acid sequences can be separatedby a linker, for example.

A “primer” is a nucleic acid that contains a sequence complementary to aregion of a template nucleic acid strand and that primes the synthesisof a strand complementary to the template (or a portion thereof).Primers are typically, but need not be, relatively short, chemicallysynthesized oligonucleotides (typically, deoxyribonucleotides). In anamplification, e.g., a PCR amplification, a pair of primers typicallydefine the 5′ ends of the two complementary strands of the nucleic acidtarget that is amplified. By “cooperative primer,” or first nucleic acidsequence, is meant a primer attached via a linker to a second nucleicacid sequence, also referred to as a capture sequence. The secondnucleic acid sequence, or capture sequence, can hybridize to thetemplate nucleic acid downstream of the 3′ end of the primer, or firstnucleic acid sequence. By “normal primer” is meant a primer which doesnot have a capture sequence, or second nucleic acid sequence, attachedto it via a linker.

By “capture sequence,” which is also referred to herein as a “secondnucleic acid sequence” is meant a sequence which hybridizes to thetarget nucleic acid and allows the first nucleic acid sequence, orprimer sequence, to be in close proximity to the target region of thetarget nucleic acid.

“Downstream” is relative to the action of the polymerase during nucleicacid synthesis or extension. For example, when the Taq polymeraseextends a primer, it adds bases to the 3′ end of the primer and willmove towards a sequence that is “downstream from the 3′ end of theprimer.”

A “target region” is a region of a target nucleic acid that is to beamplified, detected or both.

The “Tm” (melting temperature) of a nucleic acid duplex under specifiedconditions is the temperature at which half of the nucleic acidsequences are disassociated and half are associated. As used herein,“isolated Tm” refers to the individual melting temperature of either thefirst or second nucleic acid sequence in the cooperative nucleic acidwhen not in the cooperative pair. “Effective Tm” refers to the resultingmelting temperature of either the first or second nucleic acid whenlinked together.

The term “linker” means the composition joining the first and secondnucleic acids to each other. The linker comprises at least onenon-extendable moiety, but may also comprise extendable nucleic acids,and can be any length. The linker may be connected to the 3′ end, the 5′end, or can be connected one or more bases from the end (“the middle”)of both the first and second nucleic acid sequences. The connection canbe covalent, hydrogen bonding, ionic interactions, hydrophobicinteractions, and the like. The term “non-extendable”has reference tothe inability of the native Taq polymerase to recognize a moiety andthereby continue nucleic acid synthesis. A variety of natural andmodified nucleic acid bases are recognized by the polymerase and are“extendable.” Examples of non-extendable moieties include among others,fluorophores, quenchers, polyethylene glycol, polypropylene glycol,polyethylene, polypropylene, polyamides, polyesters and others known tothose skilled in the art. In some cases, even a nucleic acid base withreverse orientation (e.g. 5′ ACGT 3′ 3′A 5′ 5′ AAGT 3′) or otherwiserendered such that the Taq polymerase could not extend through it couldbe considered “non-extendable.” The term “non-nucleic acid linker” asused herein refers to a reactive chemical group that is capable ofcovalently attaching a first nucleic acid to a second nucleic acid, ormore specifically, the primer to the capture sequence. Suitable flexiblelinkers are typically linear molecules in a chain of at least one or twoatoms, more typically an organic polymer chain of 1 to 12 carbon atoms(and/or other backbone atoms) in length. Exemplary flexible linkersinclude polyethylene glycol, polypropylene glycol, polyethylene,polypropylene, polyamides, polyesters and the like.

As used herein, “complementary” or “complementarity” refers to theability of a nucleotide in a polynucleotide molecule to form a base pairwith another nucleotide in a second polynucleotide molecule. Forexample, the sequence 5′-A-C-T-3′ is complementary to the sequence3′-T-G-A-5′. Complementarity may be partial, in which only some of thenucleotides match according to base pairing, or complete, where all thenucleotides match according to base pairing. For purposes of the presentinvention “substantially complementary” refers to 90% or greateridentity over the length of the target base pair region. Thecomplementarity can also be 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100% complementary, or any amount below or inbetween these amounts.

As used herein, “amplify, amplifying, amplifies, amplified,amplification” refers to the creation of one or more identical orcomplementary copies of the target DNA. The copies may be singlestranded or double stranded. Amplification can be part of a number ofprocesses such as extension of a primer, reverse transcription,polymerase chain reaction, nucleic acid sequencing, rolling circleamplification and the like.

As used herein, “purified” refers to a polynucleotide, for example atarget nucleic acid sequence, that has been separated from cellulardebris, for example, high molecular weight DNA, RNA and protein. Thiswould include an isolated RNA sample that would be separated fromcellular debris, including DNA. It can also mean non-native, ornon-naturally occurring nucleic acid.

As used herein, “protein,” “peptide,” and “polypeptide” are usedinterchangeably to denote an amino acid polymer or a set of two or moreinteracting or bound amino acid polymers.

As used herein, “stringency” refers to the conditions, i.e.,temperature, ionic strength, solvents, and the like, under whichhybridization between polynucleotides occurs. Hybridization being theprocess that occurs between the primer and template DNA during theannealing step of the amplification process.

A variety of additional terms are defined or otherwise characterizedherein.

Materials and Methods

The present invention relates to cooperative nucleic acids, such asprimers and probes. A cooperative nucleic acid comprises anoligonucleotide primer linked to a second oligonucleotide which iscomplementary to a region of the template downstream from the 3′ end ofthe primer (as seen in FIG. 1 for example). This second oligonucleotideserves as a capture sequence. In some embodiments, this allows primerswith low melting temperatures (“Tm”) to hybridize efficiently to thetarget.

The capture sequence holds the primer in close proximity to the templateallowing extension/amplification to occur in spite of the low Tm.However, nonspecific sequences that do not have a complementary sequenceto the capture sequence, such as primer-dimers, are not extendedefficiently. Because the capture sequence uniquely hybridizes downstreamfrom the 3′ end of the primer, the specificity of amplification isachieved in every cycle. This is in contrast with conventional hot startmethods, whose specificity wears off after the first cycle.

This is also in contrast with concepts such as the dual specificityprimer (US Patent Publication 20120135473, herein incorporated byreference in its entirety for its teaching concerning dual specificityprimers). The dual specificity primer has a capture sequence linked to ashort primer via Inosine residues where the capture sequence hybridizesto the target on the 5′ side of the primer. The result is that the dualspecificity primer is highly specific in the first round ofamplification. However, if the dual specificity primers amplify eachother, the polymerase extends all the way through to the 5′ end,creating a high Tm primer-dimer that will be propagated in every roundthereafter. This is in contrast to the cooperative nucleic acid wherethe capture sequence hybridizes to the target on the 3′ side of theprimer, preventing it from being incorporated into the primer-dimer inthe order necessary to allow for propagation of the primer-dimer.

The cooperative nucleic acids and methods of using them are alsodifferent than “padlock probes” (Nilsson et al. 1994: “Padlock probes:circularizing oligonucleotides for localized DNA detection”. Science 265(5181): 2085-2088), Molecular Inversion Probes (MIPs) (Hardenbol et al2003: “Multiplexed genotyping with sequence-tagged molecular inversionprobes”. Nat Biotechnol 21 (6): 673-678) and Connector Inversion Probes(CIPs) (Akhras et al. 2007: Hall, Neil. ed. “Connector inversion probetechnology: a powerful one-primer multiplex DNA amplification system fornumerous scientific applications”. PLoS ONE 2 (9): e195). For example,the probes disclosed herein can have a linker with at least onenon-extendable moiety. Furthermore, the molecule disclosed herein is aprimer, whereas the “padlock probes” are ligated, and the non-ligatedpadlock probes are digested or otherwise removed prior to amplificationand cannot be used as primers.

Padlock probes are single stranded DNA molecules with two 20-nucleotidelong segments complementary to the target connected by a 40-nucleotidelong linker sequence. When the target complementary regions arehybridized to the DNA target, the padlock probes also becomecircularized. However, unlike MIP, padlock probes are designed such thatthe target complementary regions span the entire target region uponhybridization, leaving no gaps. Thus, padlock probes are only useful fordetecting DNA molecules with known sequences.

Molecular Inversion probes were developed to perform SNP genotyping,which are modified padlock probes such that when the probe is hybridizedto the genomic target, there is a gap at the SNP position. Gap fillingusing a nucleotide that is complementary to the nucleotide at the SNPlocation determines the identity of the polymorphism. This design bringsnumerous benefits over the more traditional padlock probe technique.Using multiple padlock probes specific to a plausible SNP requirescareful balancing of the concentration of these allele specific probesto ensure SNP counts at a given locus are properly normalized.

Connector Inversion Probes make use of a modified design of MIP byextending the gap delimited by the hybridized probe ends and named thedesign Connector Inversion Probe (CIP). The gap corresponds to thegenomic region of interest to be captured (e.g. exons). Gap fillingreaction is achieved with DNA polymerase, using all four nucleotides.Identification of the captured regions can then be done by sequencingthem using locus-specific primers that map to one of the targetcomplementary ends of the probes.

A “primer dimer” (PD) is a potential by-product in PCR. As its nameimplies, a PD consists of primer molecules that have attached(hybridized) to each other because of strings of complementary bases inthe primers or through other nonspecific interactions. As a result, theDNA polymerase amplifies the PD, leading to competition for PCRreagents, thus potentially inhibiting amplification of the DNA sequencetargeted for PCR amplification. In real-time PCR, PDs may interfere withaccurate quantification through signal dampening, false negatives, falsepositives and the like.

The present invention also relates to cooperatively linked nucleic acidsthat also comprise a probe. This modified primer/probe is similar to thecooperative nucleic acid, but with the addition of one or moredetectable labels to either the capture sequence or the primer, turningit into a probe. Because extension of the cooperative primer/probe isdetectable, it can be useful in a variety of applications includingmultiplexing applications that require differentiation of SNP's using anARMS based approach. In some embodiments, both the primer and the probeare designed with Tm's below the melting temperature which is used inthe amplification reaction, so that the primer will not amplify withoutthe probe binding and the probe will not have a signal without theprimer binding. This creates two points of specificity in the sameprimer/probe combination.

The cooperative nucleic acids, such as primers and probes, of thisinvention are useful in a variety of primer extension/amplificationreactions known to those skilled in the art, including, but not limitedto the polymerase chain reaction, rolling circle amplification, nucleicacid sequencing and others. The cooperative primers and probes of thisinvention can also be used in applications that have postextension/amplification steps, such as hybridization to an array.Because the cooperative primers/probes in this invention substantiallyreduce primer-dimers, they are of particular use in multiplexed andhighly multiplexed reactions.

The use of a cooperative nucleic acid can decrease the amount ofprimer-dimer present by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percentcompared to the amount of primer-dimer present when a normal primer (anon-cooperative nucleic acid) is used.

Therefore, disclosed herein is a cooperative nucleic acid moleculecomprising: a) a first nucleic acid sequence, wherein the first nucleicacid sequence is substantially complementary to a first region of atarget nucleic acid, and wherein the first nucleic acid sequence isextendable on the 3′ end; b) a second nucleic acid sequence, wherein thesecond nucleic acid sequence is substantially complementary to a secondregion of the target nucleic acid; wherein the first and second nucleicacid sequences are attached to each other; and wherein the secondnucleic acid sequence hybridizes to the target nucleic acid sequencedownstream from the 3′ end of the first nucleic acid sequence; andwherein the effective melting temperature (Tm) of the first nucleic acidmolecule is increased by at least 1° C. as compared to the isolated Tmof the first nucleic acid sequence without the second nucleic acidsequence attached to it.

The cooperative nucleic acid may be linear or circularized.

By “extendable on the 3′ end” is meant that the first nucleic acid isfree on this end to be amplified, or extended. This is meant to includeheat activatable primers such as those described by Lebedev et al, amongother technologies.

The first nucleic acid sequence is a primer, and the second nucleic acidsequence is alternatively referred to as a “capture nucleic acidsequence.” Either the first or the second sequence may have a detectablelabel, or a third sequence may have a detectable label. The first andsecond nucleic acid sequences can be attached via a linker, which can bea non-nucleic acid sequence. In one example, the linker can attach the5′ end of the first nucleic acid sequence to the 3′ end of the secondnucleic acid sequence. This can be seen, for instance, in FIG. 2.Alternatively, the the first nucleic acid sequence is inverted such thatthe 5′ end of the first nucleic acid sequence is attached to the 5′ endof the second nucleic acid sequence. This can be seen, for instance, inFIG. 3. In yet another example, the 5′ end of the second nucleic acidsequence can be linked to the first nucleic acid sequence in the middleof the sequence, as seen in FIG. 1. It is noted that by “middle of thesequence” is meant that the linker is not joined to the first nucleicacid sequence at either the 5′ end or the 3′ end of the nucleic acid,but rather is attached to a nucleotide internal to the nucleotides onthe 5′ and 3′ ends.

In one example, the cooperative nucleic acid comprises 75, 70, 65, 60,55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or less continuous nucleotidesin the same orientation. In other words, this is the number ofnucleotides that are part of a single, unbroken nucleic acid sequenceand oriented in the same 5′ to 3′ direction, or the 3′ to 5′ direction.By way of example, if the linker is a nucleic acid sequence, it caninclude the linker, if the nucleotides in the linker are in the sameorientation as either the first or second nucleic acid sequence to whichit is directly connected.

The linker can be made of nucleic acids, non-nucleic acids, or somecombination of both. If the linker is made of nucleic acids, it can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or 100or more nucleotides in length, or any number in between. Types oflinkers are discussed elsewhere herein. The linker can be any length,and can be longer or shorter than the combined length of the first andsecond nucleic acid sequences, longer or shorter than just the firstnucleic acid sequence, or longer or shorter than the second nucleic acidsequence.

Furthermore, there can be a space on the target nucleic acid where thefirst nucleic acid sequence and the second nucleic acid sequencehybridize. In other words, there are two distinct regions on the targetnucleic acid, one which hybridizes with the first nucleic acid sequence,and the other which hybridizes to the second nucleic acid sequence. Thedistance between the first and second regions on the target can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or 100 or morenucleotides in length.

Also disclosed herein is a kit comprising the cooperative nucleic acidmolecules disclosed herein together with instructions for their use. Insome embodiments, additional cooperative nucleic acid molecules areprovided in the kit. In still others, reagents for performing theextension are included, such as polymerase, dNTP's, buffers and thelike. In some embodiments, positive and negative controls may beincluded. In such embodiments, the reagents may all be packagedseparately or combined in a single tube or container.

Further disclosed is a method for amplifying a target nucleic acid, themethod comprising: a) providing a cooperative nucleic acid molecule asdisclosed herein; b) providing a target nucleic acid; and c) amplifyingthe target nucleic acid under appropriate conditions for amplification;wherein the effective Tm of the first nucleic acid molecule is increasedby at least 1° C. as compared to the isolated Tm of the first nucleicacid sequence without the second nucleic acid sequence attached to it;thereby amplifying the target nucleic acid.

Methods of amplification are disclosed elsewhere herein. More than onecooperative nucleic acid molecule can be provided, and they can have thesame or different sequences. For example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 60, 70, 80, 90, or 100 or more nucleic acid molecules ofdifferent sequences can be provided.

Primer Design

In some embodiments, the isolated melting temperature “Tm” of theprimer, also referred to herein as the first nucleic acid sequence, is1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more degrees belowthe reaction temperature used during the annealing phase, of PCR, or theextension phase of reactions with no annealing phase. Therefore, themelting temperature of the primer sequence can be between about 1° C.and 40° C., between about 3° C. and 20° C., between about 5° C. and 15°C. below the reaction temperature used in the PCR reaction. In apreferred embodiment, the isolated Tm is between about 7° C. and 12° C.below the reaction temperature. This provides for less than 50%, andmore preferably less than 20% of the template to be hybridized to anisolated primer.

One of skill in the art can design primers with a given meltingtemperature based on many factors, such as length, and with increasingGC content. A simple formula for calculation of the (Tm) is:

Tm=4(G+C)+2(A+T) ° C.

Furthermore, one of skill in the art will appreciate that the actual Tmis influenced by the concentration of Mg²⁺, K⁺, and cosolvents. Thereare numerous computer programs to assist in primer design.

To achieve the desired melting temperatures, the first nucleic acidsequence, or the primer, can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, and 40 bases in length. For example, theprimers can be between about 5 and 26, between about 7 and 22, betweenabout 9 and 17 bases in length depending on GC content.

Any desired number of primers of different nucleotide sequence can beused, but use of one or a few primers is preferred. The amplificationreaction can be performed with, for example, one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or seventeen primers. More primers can be used. Thereis no fundamental upper limit to the number of primers that can be used.However, the use of fewer primers is preferred. When multiple primersare used, the primers should each have a different specific nucleotidesequence.

The amplification reaction can be performed with a single primer and,for example, with no additional primers, with 1 additional primer, with2 additional primers, with 3 additional primers, with 4 additionalprimers, with 5 additional primers, with 6 additional primers, with 7additional primers, with 8 additional primers, with 9 additionalprimers, with 10 additional primers, with 11 additional primers, with 12additional primers, with 13 additional primers, with 14 additionalprimers, with 15 additional primers, with 16 additional primers, with 17additional primers, with 18 additional primers, with 19 additionalprimers, with 20 additional primers, with 21 additional primers, with 22additional primers, with 23 additional primers, with 24 additionalprimers, with 25 additional primers, with 26 additional primers, with 27additional primers, with 28 additional primers, with 29 additionalprimers, with 30 additional primers, with 31 additional primers, with 32additional primers, with 33 additional primers, with 34 additionalprimers, with 35 additional primers, with 36 additional primers, with 37additional primers, with 38 additional primers, with 39 additionalprimers, with 40 additional primers, with 41 additional primers, with 42additional primers, with 43 additional primers, with 44 additionalprimers, with 45 additional primers, with 46 additional primers, with 47additional primers, with 48 additional primers, with 49 additionalprimers, with 50 additional primers, with 51 additional primers, with 52additional primers, with 53 additional primers, with 54 additionalprimers, with 55 additional primers, with 56 additional primers, with 57additional primers, with 58 additional primers, with 59 additionalprimers, with 60 additional primers, with 61 additional primers, with 62additional primers, with 63 additional primers, with 64 additionalprimers, with 65 additional primers, with 66 additional primers, with 67additional primers, with 68 additional primers, with 69 additionalprimers, with 70 additional primers, with 71 additional primers, with 72additional primers, with 73 additional primers, with 74 additionalprimers, with 75 additional primers, with 76 additional primers, with 77additional primers, with 78 additional primers, with 79 additionalprimers, with 80 additional primers, with 81 additional primers, with 82additional primers, with 83 additional primers, with 84 additionalprimers, with 85 additional primers, with 86 additional primers, with 87additional primers, with 88 additional primers, with 89 additionalprimers, with 90 additional primers, with 91 additional primers, with 92additional primers, with 93 additional primers, with 94 additionalprimers, with 95 additional primers, with 96 additional primers, with 97additional primers, with 98 additional primers, with 99 additionalprimers, with 100 additional primers, with 110 additional primers, with120 additional primers, with 130 additional primers, with 140 additionalprimers, with 150 additional primers, with 160 additional primers, with170 additional primers, with 180 additional primers, with 190 additionalprimers, with 200 additional primers, with 210 additional primers, with220 additional primers, with 230 additional primers, with 240 additionalprimers, with 250 additional primers, with 260 additional primers, with270 additional primers, with 280 additional primers, with 290 additionalprimers, with 300 additional primers, with 310 additional primers, with320 additional primers, with 330 additional primers, with 340 additionalprimers, with 350 additional primers, with 360 additional primers, with370 additional primers, with 380 additional primers, with 390 additionalprimers, with 400 additional primers, with 410 additional primers, with420 additional primers, with 430 additional primers, with 440 additionalprimers, with 450 additional primers, with 460 additional primers, with470 additional primers, with 480 additional primers, with 490 additionalprimers, with 500 additional primers, with 550 additional primers, with600 additional primers, with 650 additional primers, with 700 additionalprimers, with 750 additional primers, with 800 additional primers, with850 additional primers, with 900 additional primers, with 950 additionalprimers, with 1,000 additional primers, with 1,100 additional primers,with 1,200 additional primers, with 1,300 additional primers, with 1,400additional primers, with 1,500 additional primers, with 1,600 additionalprimers, with 1,700 additional primers, with 1,800 additional primers,with 1,900 additional primers, with 2,000 additional primers, with 2,100additional primers, with 2,200 additional primers, with 2,300 additionalprimers, with 2,400 additional primers, with 2,500 additional primers,with 2,600 additional primers, with 2,700 additional primers, with 2,800additional primers, with 2,900 additional primers, with 3,000 additionalprimers, with 3,500 additional primers, or with 4,000 additionalprimers.

The amplification reaction can be performed with a single primer and,for example, with no additional primers, with fewer than 2 additionalprimers, with fewer than 3 additional primers, with fewer than 4additional primers, with fewer than 5 additional primers, with fewerthan 6 additional primers, with fewer than 7 additional primers, withfewer than 8 additional primers, with fewer than 9 additional primers,with fewer than 10 additional primers, with fewer than 11 additionalprimers, with fewer than 12 additional primers, with fewer than 13additional primers, with fewer than 14 additional primers, with fewerthan 15 additional primers, with fewer than 16 additional primers, withfewer than 17 additional primers, with fewer than 18 additional primers,with fewer than 19 additional primers, with fewer than 20 additionalprimers, with fewer than 21 additional primers, with fewer than 22additional primers, with fewer than 23 additional primers, with fewerthan 24 additional primers, with fewer than 25 additional primers, withfewer than 26 additional primers, with fewer than 27 additional primers,with fewer than 28 additional primers, with fewer than 29 additionalprimers, with fewer than 30 additional primers, with fewer than 31additional primers, with fewer than 32 additional primers, with fewerthan 33 additional primers, with fewer than 34 additional primers, withfewer than 35 additional primers, with fewer than 36 additional primers,with fewer than 37 additional primers, with fewer than 38 additionalprimers, with fewer than 39 additional primers, with fewer than 40additional primers, with fewer than 41 additional primers, with fewerthan 42 additional primers, with fewer than 43 additional primers, withfewer than 44 additional primers, with fewer than 45 additional primers,with fewer than 46 additional primers, with fewer than 47 additionalprimers, with fewer than 48 additional primers, with fewer than 49additional primers, with fewer than 50 additional primers, with fewerthan 51 additional primers, with fewer than 52 additional primers, withfewer than 53 additional primers, with fewer than 54 additional primers,with fewer than 55 additional primers, with fewer than 56 additionalprimers, with fewer than 57 additional primers, with fewer than 58additional primers, with fewer than 59 additional primers, with fewerthan 60 additional primers, with fewer than 61 additional primers, withfewer than 62 additional primers, with fewer than 63 additional primers,with fewer than 64 additional primers, with fewer than 65 additionalprimers, with fewer than 66 additional primers, with fewer than 67additional primers, with fewer than 68 additional primers, with fewerthan 69 additional primers, with fewer than 70 additional primers, withfewer than 71 additional primers, with fewer than 72 additional primers,with fewer than 73 additional primers, with fewer than 74 additionalprimers, with fewer than 75 additional primers, with fewer than 76additional primers, with fewer than 77 additional primers, with fewerthan 78 additional primers, with fewer than 79 additional primers, withfewer than 80 additional primers, with fewer than 81 additional primers,with fewer than 82 additional primers, with fewer than 83 additionalprimers, with fewer than 84 additional primers, with fewer than 85additional primers, with fewer than 86 additional primers, with fewerthan 87 additional primers, with fewer than 88 additional primers, withfewer than 89 additional primers, with fewer than 90 additional primers,with fewer than 91 additional primers, with fewer than 92 additionalprimers, with fewer than 93 additional primers, with fewer than 94additional primers, with fewer than 95 additional primers, with fewerthan 96 additional primers, with fewer than 97 additional primers, withfewer than 98 additional primers, with fewer than 99 additional primers,with fewer than 100 additional primers, with fewer than 110 additionalprimers, with fewer than 120 additional primers, with fewer than 130additional primers, with fewer than 140 additional primers, with fewerthan 150 additional primers, with fewer than 160 additional primers,with fewer than 170 additional primers, with fewer than 180 additionalprimers, with fewer than 190 additional primers, with fewer than 200additional primers, with fewer than 210 additional primers, with fewerthan 220 additional primers, with fewer than 230 additional primers,with fewer than 240 additional primers, with fewer than 250 additionalprimers, with fewer than 260 additional primers, with fewer than 270additional primers, with fewer than 280 additional primers, with fewerthan 290 additional primers, with fewer than 300 additional primers,with fewer than 310 additional primers, with fewer than 320 additionalprimers, with fewer than 330 additional primers, with fewer than 340additional primers, with fewer than 350 additional primers, with fewerthan 360 additional primers, with fewer than 370 additional primers,with fewer than 380 additional primers, with fewer than 390 additionalprimers, with fewer than 400 additional primers, with fewer than 410additional primers, with fewer than 420 additional primers, with fewerthan 430 additional primers, with fewer than 440 additional primers,with fewer than 450 additional primers, with fewer than 460 additionalprimers, with fewer than 470 additional primers, with fewer than 480additional primers, with fewer than 490 additional primers, with fewerthan 500 additional primers, with fewer than 550 additional primers,with fewer than 600 additional primers, with fewer than 650 additionalprimers, with fewer than 700 additional primers, with fewer than 750additional primers, with fewer than 800 additional primers, with fewerthan 850 additional primers, with fewer than 900 additional primers,with fewer than 950 additional primers, with fewer than 1,000 additionalprimers, with fewer than 1,100 additional primers, with fewer than 1,200additional primers, with fewer than 1,300 additional primers, with fewerthan fewer than 1,400 additional primers, with fewer than 1,500additional primers, with fewer than 1,600 additional primers, with fewerthan 1,700 additional primers, with fewer than 1,800 additional primers,with fewer than 1,900 additional primers, with fewer than 2,000additional primers, with fewer than 2,100 additional primers, with fewerthan 2,200 additional primers, with fewer than 2,300 additional primers,with fewer than 2,400 additional primers, with fewer than 2,500additional primers, with fewer than 2,600 additional primers, with fewerthan 2,700 additional primers, with fewer than 2,800 additional primers,with fewer than 2,900 additional primers, with fewer than 3,000additional primers, with fewer than 3,500 additional primers, or withfewer than 4,000 additional primers.

The amplification reaction can be performed, for example, with fewerthan 2 primers, with fewer than 3 primers, with fewer than 4 primers,with fewer than 5 primers, with fewer than 6 primers, with fewer than 7primers, with fewer than 8 primers, with fewer than 9 primers, withfewer than 10 primers, with fewer than 11 primers, with fewer than 12primers, with fewer than 13 primers, with fewer than 14 primers, withfewer than 15 primers, with fewer than 16 primers, with fewer than 17primers, with fewer than 18 primers, with fewer than 19 primers, withfewer than 20 primers, with fewer than 21 primers, with fewer than 22primers, with fewer than 23 primers, with fewer than 24 primers, withfewer than 25 primers, with fewer than 26 primers, with fewer than 27primers, with fewer than 28 primers, with fewer than 29 primers, withfewer than 30 primers, with fewer than 31 primers, with fewer than 32primers, with fewer than 33 primers, with fewer than 34 primers, withfewer than 35 primers, with fewer than 36 primers, with fewer than 37primers, with fewer than 38 primers, with fewer than 39 primers, withfewer than 40 primers, with fewer than 41 primers, with fewer than 42primers, with fewer than 43 primers, with fewer than 44 primers, withfewer than 45 primers, with fewer than 46 primers, with fewer than 47primers, with fewer than 48 primers, with fewer than 49 primers, withfewer than 50 primers, with fewer than 51 primers, with fewer than 52primers, with fewer than 53 primers, with fewer than 54 primers, withfewer than 55 primers, with fewer than 56 primers, with fewer than 57primers, with fewer than 58 primers, with fewer than 59 primers, withfewer than 60 primers, with fewer than 61 primers, with fewer than 62primers, with fewer than 63 primers, with fewer than 64 primers, withfewer than 65 primers, with fewer than 66 primers, with fewer than 67primers, with fewer than 68 primers, with fewer than 69 primers, withfewer than 70 primers, with fewer than 71 primers, with fewer than 72primers, with fewer than 73 primers, with fewer than 74 primers, withfewer than 75 primers, with fewer than 76 primers, with fewer than 77primers, with fewer than 78 primers, with fewer than 79 primers, withfewer than 80 primers, with fewer than 81 primers, with fewer than 82primers, with fewer than 83 primers, with fewer than 84 primers, withfewer than 85 primers, with fewer than 86 primers, with fewer than 87primers, with fewer than 88 primers, with fewer than 89 primers, withfewer than 90 primers, with fewer than 91 primers, with fewer than 92primers, with fewer than 93 primers, with fewer than 94 primers, withfewer than 95 primers, with fewer than 96 primers, with fewer than 97primers, with fewer than 98 primers, with fewer than 99 primers, withfewer than 100 primers, with fewer than 110 primers, with fewer than 120primers, with fewer than 130 primers, with fewer than 140 primers, withfewer than 150 primers, with fewer than 160 primers, with fewer than 170primers, with fewer than 180 primers, with fewer than 190 primers, withfewer than 200 primers, with fewer than 210 primers, with fewer than 220primers, with fewer than 230 primers, with fewer than 240 primers, withfewer than 250 primers, with fewer than 260 primers, with fewer than 270primers, with fewer than 280 primers, with fewer than 290 primers, withfewer than 300 primers, with fewer than 310 primers, with fewer than 320primers, with fewer than 330 primers, with fewer than 340 primers, withfewer than 350 primers, with fewer than 360 primers, with fewer than 370primers, with fewer than 380 primers, with fewer than 390 primers, withfewer than 400 primers, with fewer than 410 primers, with fewer than 420primers, with fewer than 430 primers, with fewer than 440 primers, withfewer than 450 primers, with fewer than 460 primers, with fewer than 470primers, with fewer than 480 primers, with fewer than 490 primers, withfewer than 500 primers, with fewer than 550 primers, with fewer than 600primers, with fewer than 650 primers, with fewer than 700 primers, withfewer than 750 primers, with fewer than 800 primers, with fewer than 850primers, with fewer than 900 primers, with fewer than 950 primers, withfewer than 1,000 primers, with fewer than 1,100 primers, with fewer than1,200 primers, with fewer than 1,300 primers, with fewer than fewer than1,400 primers, with fewer than 1,500 primers, with fewer than 1,600primers, with fewer than 1,700 primers, with fewer than 1,800 primers,with fewer than 1,900 primers, with fewer than 2,000 primers, with fewerthan 2,100 primers, with fewer than 2,200 primers, with fewer than 2,300primers, with fewer than 2,400 primers, with fewer than 2,500 primers,with fewer than 2,600 primers, with fewer than 2,700 primers, with fewerthan 2,800 primers, with fewer than 2,900 primers, with fewer than 3,000primers, with fewer than 3,500 primers, or with fewer than 4,000primers.

The disclosed primers can have one or more modified nucleotides. Suchprimers are referred to herein as modified primers. Chimeric primers canalso be used. Chimeric primers are primers having at least two types ofnucleotides, such as both deoxyribonucleotides and ribonucleotides,ribonucleotides and modified nucleotides, two or more types of modifiednucleotides, deoxyribonucleotides and two or more different types ofmodified nucleotides, ribonucleotides and two or more different types ofmodified nucleotides, or deoxyribonucleotides, ribonucleotides and twoor more different types of modified nucleotides. One form of chimericprimer is peptide nucleic acid/nucleic acid primers. For example,5′-PNA-DNA-3′ or 5′-PNA-RNA-3′ primers may be used for more efficientstrand invasion and polymerization invasion. Other forms of chimericprimers are, for example, 5′-(2′-O-Methyl) RNA-RNA-3′ or5′-(2′-O-Methyl) RNA-DNA-3′.

Many modified nucleotides (nucleotide analogs) are known and can be usedin oligonucleotides. A nucleotide analog is a nucleotide which containssome type of modification to either the base, sugar, or phosphatemoieties. Modifications to the base moiety would include natural andsynthetic modifications of A, C, G, and T/U as well as different purineor pyrimidine bases, such as uracil-5-yl, hypoxanthin-9-yl (I), and2-aminoadenin-9-yl. A modified base includes but is not limited to5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional basemodifications can be found for example in U.S. Pat. No. 3,687,808,Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and Sanghvi, Y. S., Chapter 15, Antisense Research andApplications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRCPress, 1993. Certain nucleotide analogs, such as 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine can increase the stability of duplex formation. Othermodified bases are those that function as universal bases. Universalbases include 3-nitropyrrole and 5-nitroindole. Universal basessubstitute for the normal bases but have no bias in base pairing. Thatis, universal bases can base pair with any other base. A primer havingone or more universal bases is not considered to be a primer having aspecific sequence.

Base modifications often can be combined with for example a sugarmodification, such as 2′-O-methoxyethyl, to achieve unique propertiessuch as increased duplex stability. There are numerous United Statespatents such as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, which detail and describe a range of basemodifications. Each of these patents is herein incorporated byreference.

Nucleotide analogs can also include modifications of the sugar moiety.Modifications to the sugar moiety would include natural modifications ofthe ribose and deoxyribose as well as synthetic modifications. Sugarmodifications include but are not limited to the following modificationsat the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-,S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl may be substituted or unsubstituted C1 to C10, alkyl or C2 toC10 alkenyl and alkynyl. 2′ sugar modifications also include but are notlimited to —O[(CH₂)_(n) O]_(m) CH₃, —O(CH₂)_(n) OCH₃, —O(CH₂)_(n) NH₂,—O(CH₂)_(n) CH₃, —O(CH₂)_(n) —ONH₂, and —O(CH₂)_(n)ON[(CH₂)_(n) CH₃)]₂,where n and m are from 1 to about 10.

Other modifications at the 2′ position include but are not limited to:C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Similar modifications mayalso be made at other positions on the sugar, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide. Modifiedsugars would also include those that contain modifications at thebridging ring oxygen, such as CH₂ and S. Nucleotide sugar analogs mayalso have sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. There are numerous United States patents thatteach the preparation of such modified sugar structures such as U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference in its entirety.

Nucleotide analogs can also be modified at the phosphate moiety.Modified phosphate moieties include but are not limited to those thatcan be modified so that the linkage between two nucleotides contains aphosphorothioate, chiral phosphorothioate, phosphorodithioate,phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates including 3′-alkylene phosphonate and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates. It is understood that these phosphate or modifiedphosphate linkages between two nucleotides can be through a 3′-5′linkage or a 2′-5′ linkage, and the linkage can contain invertedpolarity such as 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixedsalts and free acid forms are also included. Numerous United Statespatents teach how to make and use nucleotides containing modifiedphosphates and include but are not limited to, U.S. Pat. Nos. 3,687,808;4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050,each of which is herein incorporated by reference.

It is understood that nucleotide analogs need only contain a singlemodification, but may also contain multiple modifications within one ofthe moieties or between different moieties.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize and hybridize to complementary nucleic acids in aWatson-Crick or Hoogsteen manner, but which are linked together througha moiety other than a phosphate moiety. Nucleotide substitutes are ableto conform to a double helix type structure when interacting with theappropriate nucleic acid molecules.

Nucleotide substitutes are nucleotides or nucleotide analogs that havehad the phosphate moiety and/or sugar moieties replaced. Nucleotidesubstitutes do not contain a standard phosphorus atom. Substitutes forthe phosphate can be for example, short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts. Numerous United States patents disclosehow to make and use these types of phosphate replacements and includebut are not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439,each of which is herein incorporated by reference.

It is also understood in a nucleotide substitute that both the sugar andthe phosphate moieties of the nucleotide can be replaced, by for examplean amide type linkage (aminoethylglycine) (PNA). U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNAmolecules, each of which is herein incorporated by reference. (See alsoNielsen et al., Science 254:1497-1500 (1991)).

Primers can be comprised of nucleotides and can be made up of differenttypes of nucleotides or the same type of nucleotides. For example, oneor more of the nucleotides in a primer can be ribonucleotides,2′-O-methyl ribonucleotides, or a mixture of ribonucleotides and2′-O-methyl ribonucleotides; about 10% to about 50% of the nucleotidescan be ribonucleotides, 2′-O-methyl ribonucleotides, or a mixture ofribonucleotides and 2′-O-methyl ribonucleotides; about 50% or more ofthe nucleotides can be ribonucleotides, 2′-O-methyl ribonucleotides, ora mixture of ribonucleotides and 2′-O-methyl ribonucleotides; or all ofthe nucleotides are ribonucleotides, 2′-O-methyl ribonucleotides, or amixture of ribonucleotides and 2′-O-methyl ribonucleotides. Thenucleotides can be comprised of bases (that is, the base portion of thenucleotide) and can (and normally will) comprise different types ofbases.

Capture Sequence Design

The capture sequence, also referred to herein as the “second nucleicacid sequence,” is complementary to the template such that it hybridizesto the target nucleic acid molecule downstream from the 3′ end of theprimer. In some embodiments, resistance to mutations in the targetnucleic acid is desired and the capture sequence is designed with amelting temperature greater than the reaction temperature. In theseembodiments, the capture sequence is designed with an isolated Tm of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more degrees above thereaction temperature. For example, the capture, or second, sequence isbetween about 0° C. and 40° C., between about 5° C. and 30° C., betweenabout 7° C. and 25° C. above the reaction temperature. In someembodiments, the predicted melting temperature of the capture sequenceis also made for expected mutants. In these embodiments, the isolated Tmof the capture sequence to the expected mutants is between about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, 19, 20, or moredegrees C. below the reaction temperature, or 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50or more degrees C. above the reaction temperature. For example, it canbe 10° C. below the reaction temperature and 30° C. above the reactiontemperature, between about 3° C. below the reaction temperature andabout 10° C. above the reaction temperature.

To achieve these melting temperatures, the capture sequence length canbe 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 ormore bases in length. For example, it can be between about 20 and about50, between about 22 and about 40, between about 23 and about 37 bases.

In some embodiments, an even higher resistance to mutations in thetarget sequence is desired. In these embodiments, in addition to acapture sequence with an isolated Tm of between about 0° C. and 40° C.above the reaction temperature, the cooperative primer is designed withan isolated Tm of between about 7° C. below and about 20° C. above,between about 5° C. below and about 10° C. above, between about 3° C.below and about 3° C. above the reaction temperature. The cooperativeinteraction between the primer and the capture sequence will result inan even greater effective Tm for the cooperative primer, rendering italmost impervious to mutations in the sequence. By comparison, a normalprimer might have to be an additional 5 to 30 bases in length to have anequivalent resistance to mutations in the target sequence, andconsequently, would be much more susceptible to primer-dimer formation.

In other embodiments, a higher resistance to primer-dimers is preferredand the melting temperature of the isolated capture, or second, nucleicacid sequence is designed to be less than the reaction temperature. Forexample, the capture, or second, nucleic acid sequence is 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50 or more degrees below the reactiontemperature, or annealing phase, of PCR. In preferred embodiments, theTm of the isolated capture, or second nucleic acid, sequence is betweenabout 0° C. and 12° C., between about 1° C. and 8° C., between about 2°C. and 5° C. below the reaction temperature. To achieve these lowmelting temperatures, the capture sequence length can be 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, or 50 or more bases in length. For example, thecapture, or second nucleic acid sequence, can be between about 5 and 30,between about 8 and 25, and between about 10 and 22 bases.

In some embodiments, the capture sequence binds and releases the targetsequence rapidly such that the polymerase can extend underneath thecapture sequence, leaving the capture sequence intact. In someembodiments, this is enhanced using a cooperative primer with the linkerattached to the 5′ end of the capture sequence. In a preferredembodiment, the polymerase is capable of cleaving the capture sequenceduring extension. In a preferred embodiment, this is enhanced using acooperative primer with the linker attached to the 3′ end of the capturesequence.

Linker

The number of bases between the 3′ end of the first nucleic acid, orprimer, sequence and the 5′ end of the second nucleic acid, or capturesequence hybridization locations in the template is important. In someembodiments, the number of bases between the primer and the capturesequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. Forexample, they can be between about 0 and 30, between about 0 and 20,between about 0 and 10 bases.

The more bases that are between the two sites, the longer the linkerneeds to be if cleavage of the capture sequence is desired. The longerthe linker, the more entropy that enters into the system, which lowersthe effect of cooperative binding. This is expressed in the followingequation:

K _(eff) =K _(primer) +K _(capture) +L _(C) K _(primer) K _(capture)

Where Keff is the effective or cooperative equilibrium constant, Kprimeris the equilibrium constant of the primer in isolation, Kcapture is theequilibrium constant of the capture sequence in equilibrium and Lc isthe local concentration defined as:

$L_{c} = \frac{\left( \frac{1}{6.022E\; 23} \right)}{\frac{4}{3}\pi \; r^{3}}$

Where r is the linker length in decimeters. This provides the effectivelocal concentration in molarity due to the cooperative interactionbetween the primer and the probe. Accordingly, linker length directlydetermines the cooperative contribution (L_(c)K_(primer)K_(capture)) tothe effective equilibrium constant.

K_(primer) and K_(capture) can be calculated by obtaining the enthalpyand entropy values for the primer and the capture sequences usingnearest neighbor or other calculations known to those skilled in theart.

The total amount of template bound by the primer can be calculated asfollows:

$\frac{T_{primer}}{T_{o}} = \frac{\left( {K_{primer} + {L_{C}K_{primer}K_{capture}}} \right)P_{o}}{1 + {\left( {K_{primer} + K_{capture} + {L_{c}K_{primer}K_{capture}}} \right)P_{o}}}$

Where Tprimer is the template bound by primer, T_(o) is the total amountof template and P_(o) is the starting cooperative primer concentration.It can be seen that the cooperative effect is greatest whenL_(c)K_(primer)K_(capture) is much greater than K_(primer). For this tooccur the linker length should be as short as possible.

While the math shows that the linker length should be as short aspossible, there are several limitations to how short the linker canactually be. When the capture sequence and the probe bind to thetemplate, they form rigid double helices. The linker length must besufficient to accommodate this structure.

In some embodiments, the linker attaches the 5′ end of the primer to the3′ end of the capture sequence (FIG. 2). In this embodiment, the linkeris larger than the combined length of the primer and capture sequences.In a preferred embodiment where the linker attaches to the 3′ end of thecapture sequence, the linker comprises 6 hexaethylene glycols. Inanother embodiment, the primer is inverted such that the 5′ end of theprimer is attached to the 5′ end of the capture sequence (FIG. 3). Inthis embodiment, the linker is longer than the primer. In a preferredembodiment where the linker attaches to the 5′ end of the capturesequence, the linker comprises 3 hexaethylene glycols. In yet anotherembodiment, the 3′ end of the capture sequence is linked to the middleof the primer (FIG. 1). In this instance, the linker may be shorter thanthe length of the primer.

A variety of linker types and compositions are known to those skilled inthe art. Examples include, but are not limited to, polyethylene glycoland carbon linkers. Linkers can be attached through a variety ofmethods, including but not limited to, covalent bonds, ionic bonds,hydrogen bonding, polar association, magnetic association, and van derwals association. A preferred method is covalent bonding throughstandard DNA synthesis methods.

The length of polyethylene glycol linkers is about 0.34 nm per monomer.In some embodiments, the length of the polyethylene glycol linker isbetween about 1 and 90, between about 2 and 50, between about 3 and 30monomers (between about 1 and 10 nm fully extended).

Using the Capture Sequence as a Probe

In some embodiments, it is preferable to have the capture sequence alsoserve as a probe. In some embodiments, this is done through the additionof one or more labels to the capture sequence. In a preferredembodiment, the labels include a FRET pair.

Various nucleic acid probe constructs are known to those skilled in theart. These include, but are not limited to, dual labeled probes, hairpinprobes, and single label probes (see FIG. 4).

In some embodiments, a low background signal is desired for high signalto noise ratios. In some embodiments, a hairpin probe is used to provideincreased contact quenching to assist in providing high signal to noise.

In other embodiments, a shorter probe is desired to minimizeprimer-probe dimers. In some embodiments requiring a shorter probe, adual labeled probe is used. In embodiments that require an even greateremphasis on the reduction of spurious extension products, the meltingtemperature of the isolated probe target complex is less than thereaction temperature.

A variety of methods for detecting signal from labeled probes are knownto those skilled in the art. In some embodiments a polymerase is usedthat cleaves the probe, releasing a label that changes the signal. Inother embodiments, a polymerase is used that does not cleave the probe.Rather the signal is modified by the hybridization of the probe to thetemplate.

Using a Primer with a Built in Detection Mechanism

In some embodiments, the primer has a built in detection mechanism. Insome embodiments the detection mechanism includes one or more detectablelabels. In a preferred embodiment, the detection mechanism includes aFRET pair. Examples of primers with built in detection mechanismsinclude, but are not limited to, Amplifluor primers, Rapid Detexprimers, and others known to those skilled in the art. An example ofthis is seen in FIG. 7.

Cooperative nucleic acids with built in detection mechanisms can be moreuseful to assay designers than non-cooperative nucleic acids (normalprimers) with built in detection mechanisms. Without being limited bytheory, this is because cooperative nucleic acids are less prone togenerate signal from nonspecific products, such as primer-dimers.

In some embodiments, a nucleic acid binding dye, such as SYBR Green, isused to monitor the progress of the amplification reaction.

Fluorescent change probes and fluorescent change primers refer to allprobes and primers that involve a change in fluorescence intensity orwavelength based on a change in the form or conformation of the probe orprimer and nucleic acid to be detected, assayed or replicated. Examplesof fluorescent change probes and primers include molecular beacons,Amplifluors, FRET probes, cleavable FRET probes, TaqMan probes, scorpionprimers, fluorescent triplex oligos, fluorescent water-solubleconjugated polymers, PNA probes and QPNA probes.

Fluorescent change probes and primers can be classified according totheir structure and/or function. Fluorescent change probes includehairpin quenched probes, cleavage quenched probes, cleavage activatedprobes, and fluorescent activated probes. Fluorescent change primersinclude stem quenched primers and hairpin quenched primers. The use ofseveral types of fluorescent change probes and primers are reviewed inSchweitzer and Kingsmore, Curr. Opin. Biotech. 12:21-27 (2001). Hall etal., Proc. Natl. Acad. Sci. USA 97:8272-8277 (2000), describe the use offluorescent change probes with Invader assays.

Hairpin quenched probes are probes that when not bound to a targetsequence form a hairpin structure (and, typically, a loop) that brings afluorescent label and a quenching moiety into proximity such thatfluorescence from the label is quenched. When the probe binds to atarget sequence, the stem is disrupted, the quenching moiety is nolonger in proximity to the fluorescent label and fluorescence increases.Examples of hairpin quenched probes are molecular beacons, fluorescenttriplex oligos, and QPNA probes.

Cleavage activated probes are probes where fluorescence is increased bycleavage of the probe. Cleavage activated probes can include afluorescent label and a quenching moiety in proximity such thatfluorescence from the label is quenched. When the probe is clipped ordigested (typically by the 5′-3′ exonuclease activity of a polymeraseduring amplification), the quenching moiety is no longer in proximity tothe fluorescent label and fluorescence increases. TaqMan probes (Hollandet al., Proc. Natl. Acad. Sci. USA 88:7276-7280 (1991)) are an exampleof cleavage activated probes.

Cleavage quenched probes are probes where fluorescence is decreased oraltered by cleavage of the probe. Cleavage quenched probes can includean acceptor fluorescent label and a donor moiety such that, when theacceptor and donor are in proximity, fluorescence resonance energytransfer from the donor to the acceptor causes the acceptor tofluoresce. The probes are thus fluorescent, for example, when hybridizedto a target sequence. When the probe is clipped or digested (typicallyby the 5′-3′ exonuclease activity of a polymerase during amplification),the donor moiety is no longer in proximity to the acceptor fluorescentlabel and fluorescence from the acceptor decreases. If the donor moietyis itself a fluorescent label, it can release energy as fluorescence(typically at a different wavelength than the fluorescence of theacceptor) when not in proximity to an acceptor. The overall effect wouldthen be a reduction of acceptor fluorescence and an increase in donorfluorescence. Donor fluorescence in the case of cleavage quenched probesis equivalent to fluorescence generated by cleavage activated probeswith the acceptor being the quenching moiety and the donor being thefluorescent label. Cleavable FRET (fluorescence resonance energytransfer) probes are an example of cleavage quenched probes.

Fluorescent activated probes are probes or pairs of probes wherefluorescence is increased or altered by hybridization of the probe to atarget sequence. Fluorescent activated probes can include an acceptorfluorescent label and a donor moiety such that, when the acceptor anddonor are in proximity (when the probes are hybridized to a targetsequence), fluorescence resonance energy transfer from the donor to theacceptor causes the acceptor to fluoresce. Fluorescent activated probesare typically pairs of probes designed to hybridize to adjacentsequences such that the acceptor and donor are brought into proximity.Fluorescent activated probes can also be single probes containing both adonor and acceptor where, when the probe is not hybridized to a targetsequence, the donor and acceptor are not in proximity but where thedonor and acceptor are brought into proximity when the probe hybridizedto a target sequence. This can be accomplished, for example, by placingthe donor and acceptor on opposite ends a the probe and placing targetcomplement sequences at each end of the probe where the targetcomplement sequences are complementary to adjacent sequences in a targetsequence. If the donor moiety of a fluorescent activated probe is itselfa fluorescent label, it can release energy as fluorescence (typically ata different wavelength than the fluorescence of the acceptor) when notin proximity to an acceptor (that is, when the probes are not hybridizedto the target sequence). When the probes hybridize to a target sequence,the overall effect would then be a reduction of donor fluorescence andan increase in acceptor fluorescence. FRET probes are an example offluorescent activated probes.

Stem quenched primers are primers that when not hybridized to acomplementary sequence form a stem structure (either an intramolecularstem structure or an intermolecular stem structure) that brings afluorescent label and a quenching moiety into proximity such thatfluorescence from the label is quenched. When the primer binds to acomplementary sequence, the stem is disrupted, the quenching moiety isno longer in proximity to the fluorescent label and fluorescenceincreases. In the disclosed method, stem quenched primers are used asprimers for nucleic acid synthesis and thus become incorporated into thesynthesized or amplified nucleic acid. Examples of stem quenched primersare peptide nucleic acid quenched primers and hairpin quenched primers.

Peptide nucleic acid quenched primers are primers associated with apeptide nucleic acid quencher or a peptide nucleic acid fluor to form astem structure. The primer contains a fluorescent label or a quenchingmoiety and is associated with either a peptide nucleic acid quencher ora peptide nucleic acid fluor, respectively. This puts the fluorescentlabel in proximity to the quenching moiety. When the primer isreplicated, the peptide nucleic acid is displaced, thus allowing thefluorescent label to produce a fluorescent signal.

Hairpin quenched primers are primers that when not hybridized to acomplementary sequence form a hairpin structure (and, typically, a loop)that brings a fluorescent label and a quenching moiety into proximitysuch that fluorescence from the label is quenched. When the primer bindsto a complementary sequence, the stem is disrupted, the quenching moietyis no longer in proximity to the fluorescent label and fluorescenceincreases. Hairpin quenched primers are typically used as primers fornucleic acid synthesis and thus become incorporated into the synthesizedor amplified nucleic acid. Examples of hairpin quenched primers areAmplifluor primers (Nazerenko et al., Nucleic Acids Res. 25:2516-2521(1997)) and scorpion primers (Thelwell et al., Nucleic Acids Res.28(19):3752-3761 (2000)).

Cleavage activated primers are similar to cleavage activated probesexcept that they are primers that are incorporated into replicatedstrands and are then subsequently cleaved. Little et al., Clin. Chem.45:777-784 (1999), describe the use of cleavage activated primers.

Multiplexing with ARMS

In some embodiments, detection of multiple polymorphisms, insertions,deletions or other mutations is desired. In some embodiments, the primeris designed such that the base on the 3′ end is over the mutation. Insome embodiments, additional intentional polymorphisms are designed intothe primer. In one embodiment, the presence of a probe attached to theprimer allows for allele specific real-time detection of multiplepolymorphisms in the same location.

Mutation Differentiation with the Probe

In some embodiments the differentiation of polymorphisms is accomplishedusing the capture sequence attached to the primer. In some embodimentsthe capture sequence has additional mutations intentionally added toimprove differentiation. In some embodiments, the capture sequence willnot bind when a polymorphism is present, preventing efficientamplification round after around. In some embodiments where the capturesequence has a detectable label, even if some amplification does occur,the capture sequence does not bind sufficiently to generate a detectablesignal.

RNA and Other Reactions

In some embodiments, a polymerase other than a DNA polymerase is used. Avariety of polymerases and enzymes capable of adding one or more basesto a nucleic acid template are known to those skilled in the art. Insome embodiments, reverse transcription is desired. In some embodiments,the probe has a sufficiently low melting temperature that the polymerasecan extend underneath it. In other embodiments, an increase in thetemperature after a time for initial polymerization removes the capturesequence from the template, allowing the polymerase to extend. In otherembodiments, additional primer sequences are used that do not have acapture sequence, allowing the polymerase to make copies in anuninhibited fashion at lower reaction temperatures.

Target Nucleic Acid Molecules

Nucleic acid molecules, which are the object of amplification, can beany nucleic acid from any source. In general, the disclosed method isperformed using a nucleic acid sample that contains (or is suspected ofcontaining) nucleic acid molecules to be amplified.

A nucleic acid sample can be any nucleic acid sample of interest. Thesource, identity, and preparation of many such nucleic acid samples areknown. It is preferred that nucleic acid samples known or identified foruse in amplification or detection methods be used for the methoddescribed herein. The nucleic acid sample can be, for example, a nucleicacid sample from one or more cells, tissue, or bodily fluids such asblood, urine, semen, lymphatic fluid, cerebrospinal fluid, or amnioticfluid, or other biological samples, such as tissue culture cells, buccalswabs, mouthwash, stool, tissues slices, biopsy aspiration, andarcheological samples such as bone or mummified tissue. Types of usefulnucleic acid samples include blood samples, urine samples, semensamples, lymphatic fluid samples, cerebrospinal fluid samples, amnioticfluid samples, biopsy samples, needle aspiration biopsy samples, cancersamples, tumor samples, tissue samples, cell samples, cell lysatesamples, a crude cell lysate samples, forensic samples, archeologicalsamples, infection samples, nosocomial infection samples, productionsamples, drug preparation samples, biological molecule productionsamples, protein preparation samples, lipid preparation samples, and/orcarbohydrate preparation samples.

For whole genome amplification, preferred nucleic acid samples arenucleic acid samples from a single cell. The nucleic acid samples foruse in the disclosed method are preferably nucleic acid molecules andsamples that are complex and non-repetitive. Where the nucleic acidsample is a genomic nucleic acid sample, the genome can be the genomefrom any organism of interest. For example, the genome can be a viralgenome, a bacterial genome, a eubacterial genome, an archae bacterialgenome, a fungal genome, a microbial genome, a eukaryotic genome, aplant genome, an animal genome, a vertebrate genome, an invertebrategenome, an insect genome, a mammalian genome, or a human genome. Thetarget genome is preferably pure or substantially pure, but this is notrequired. For example, an genomic sample from an animal source mayinclude nucleic acid from contaminating or infecting organisms.

The nucleic acid sample can be, or can be derived from, for example, oneor more whole genomes from the same or different organisms, tissues,cells or a combination; one or more partial genomes from the same ordifferent organisms, tissues, cells or a combination; one or more wholechromosomes from the same or different organisms, tissues, cells or acombination; one or more partial chromosomes from the same or differentorganisms, tissues, cells or a combination; one or more chromosomefragments from the same or different organisms, tissues, cells or acombination; one or more artificial chromosomes; one or more yeastartificial chromosomes; one or more bacterial artificial chromosomes;one or more cosmids; or any combination of these.

Oligonucleotide Synthesis

Primers, detection probes, address probes, and any otheroligonucleotides can be synthesized using established oligonucleotidesynthesis methods. Methods to produce or synthesize oligonucleotides arewell known in the art. Such methods can range from standard enzymaticdigestion followed by nucleotide fragment isolation (see for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)Chapters 5, 6) to purely synthetic methods, for example, by thecyanoethyl phosphoramidite method. Solid phase chemical synthesis of DNAfragments is routinely performed using protected nucleoside cyanoethylphosphoramidites (S. L. Beaucage et al. (1981) Tetrahedron Lett.22:1859). In this approach, the 3′-hydroxyl group of an initial5′-protected nucleoside is first covalently attached to the polymersupport (R. C. Pless et al. (1975) Nucleic Acids Res. 2:773 (1975)).Synthesis of the oligonucleotide then proceeds by deprotection of the5′-hydroxyl group of the attached nucleoside, followed by coupling of anincoming nucleoside-3′-phosphoramidite to the deprotected hydroxyl group(M. D. Matteucci et a. (1981) J. Am. Chem. Soc. 103:3185). The resultingphosphite triester is finally oxidized to a phosphorotriester tocomplete the intemucleotide bond (R. L. Letsinger et al. (1976) J. Am.Chem. Soc. 9:3655). Alternatively, the synthesis of phosphorothioatelinkages can be carried out by sulfurization of the phosphite triester.Several chemicals can be used to perform this reaction, among them3H-1,2-benzodithiole-3-one, 1,1-dioxide (R. P. Iyer, W. Egan, J. B.Regan, and S. L. Beaucage, J. Am. Chem. Soc., 1990, 112, 1253-1254). Thesteps of deprotection, coupling and oxidation are repeated until anoligonucleotide of the desired length and sequence is obtained. Othermethods exist to generate oligonucleotides such as the H-phosphonatemethod (Hall et al, (1957) J. Chem. Soc., 3291-3296) or thephosphotriester method as described by Ikuta et al., Ann. Rev. Biochem.53:323-356 (1984), (phosphotriester and phosphite-triester methods), andNarang et al., Methods Enzymol., 65:610-620 (1980), (phosphotriestermethod). Protein nucleic acid molecules can be made using known methodssuch as those described by Nielsen et al., Bioconjug. Chem. 5:3-7(1994). Other forms of oligonucleotide synthesis are described in U.S.Pat. No. 6,294,664 and U.S. Pat. No. 6,291,669.

The nucleotide sequence of an oligonucleotide is generally determined bythe sequential order in which subunits of subunit blocks are added tothe oligonucleotide chain during synthesis. Each round of addition caninvolve a different, specific nucleotide precursor, or a mixture of oneor more different nucleotide precursors. For the disclosed primers ofspecific sequence, specific nucleotide precursors would be addedsequentially.

Many of the oligonucleotides described herein are designed to becomplementary to certain portions of other oligonucleotides or nucleicacids such that stable hybrids can be formed between them. The stabilityof these hybrids can be calculated using known methods such as thosedescribed in Lesnick and Freier, Biochemistry 34:10807-10815 (1995),McGraw et al., Biotechniques 8:674-678 (1990), and Rychlik et al.,Nucleic Acids Res. 18:6409-6412 (1990).

So long as their relevant function is maintained, primers, detectionprobes, address probes, and any other oligonucleotides can be made up ofor include modified nucleotides (nucleotide analogs). Many modifiednucleotides are known and can be used in oligonucleotides, and aredisclosed elsewhere herein.

Kits

The materials described above as well as other materials can be packagedtogether in any suitable combination as a kit useful for performing, oraiding in the performance of, the disclosed method. It is useful if thekit components in a given kit are designed and adapted for use togetherin the disclosed method. For example disclosed are kits foramplification of nucleic acid samples, the kit comprising cooperativenucleic acids and a DNA polymerase. The kits also can containnucleotides, buffers, detection probes, fluorescent change probes, lysissolutions, stabilization solutions, denaturation solutions, or acombination.

Uses

The disclosed method and compositions are applicable to numerous areasincluding, but not limited to, analysis of nucleic acids present incells (for example, analysis of genomic DNA in cells), diseasedetection, mutation detection, gene discovery, gene mapping (molecularhaplotyping), and agricultural research. Particularly useful is wholegenome amplification. Other uses include, for example, detection ofnucleic acids in cells and on genomic DNA arrays; molecular haplotyping;mutation detection; detection of inherited diseases such as cysticfibrosis, muscular dystrophy, diabetes, hemophilia, sickle cell anemia;assessment of predisposition for cancers such as prostate cancer, breastcancer, lung cancer, colon cancer, ovarian cancer, testicular cancer,pancreatic cancer.

Amplification

Amplification methods suitable for use with the present methods include,for example, polymerase chain reaction (PCR), reverse transcriptionPCR(RT-PCR), ligase chain reaction (LCR), transcription-basedamplification system (TAS), nucleic acid sequence based amplification(NASBA) reaction, self-sustained sequence replication (3SR), stranddisplacement amplification (SDA) reaction, boomerang DNA amplification(BDA), Q-beta replication: or isothermal nucleic acid sequence basedamplification. These methods of amplification each described brieflybelow and are well-known in the art.

PCR is a technique for making many copies of a specific template DNAsequence. The reaction consists of multiple amplification cycles and isinitiated using a pair of primer oligonucleotides that hybridize to the5′ and 3′ ends of the sequence to be copied. The amplification cycleincludes an initial denaturation, and up to 50 cycles of annealing,strand elongation (or extension) and strand separation (denaturation).In each cycle of the reaction, the DNA sequence between the primers iscopied. Primers can bind to the copied DNA as well as the originaltemplate sequence, so the total number of copies increases exponentiallywith time. PCR can be performed as according to Whelan, et al, Journalof Clinical Microbiology, 33(3):556-561 (1995). Briefly, a PCR reactionmixture includes two specific primers, dNTPs, Taq polymerase, and 1×PCRBuffer, which is amplified using a thermal cycler. Cycling parameterscan be varied, depending on, for example, the melting temperature of theprimer or the length of nucleic acids to be extended. The skilledartisan is capable of designing and preparing primers that areappropriate for amplifying a target sequence. The length of theamplification primers for use in the present invention depends onseveral factors including the nucleotide sequence identity and thetemperature at which these nucleic acids are hybridized or used duringin vitro nucleic acid amplification. The considerations necessary todetermine a preferred length for an amplification primer of a particularsequence identity are well-known to a person of ordinary skill andinclude considerations described herein. For example, the length of ashort nucleic acid or oligonucleotide can relate to its hybridizationspecificity or selectivity.

Real time PCR is PCR-based amplification method in which PCR productsare detected in real time, that is, the accumulation of PCR products canbe determined at each cycle. An example of Real Time PCR is performedusing TaqMan probes in combination with a suitableamplification/analyzer such as Applied Biosystems (ABI) Prism 7900HTSequence Detection System, which is a high-throughput real-time PCRsystem. Briefly, TaqMan probes specific for the amplified targetsequence are included in the PCR amplification reaction. These probescontain a reporter dye at the 5′ end and a quencher dye at the 3′ end.Probes hybridizing to different target sequences are conjugated with adifferent fluorescent reporter dye. In this way, more than one targetsequence can be assayed for in the same reaction vessel. During PCR, thefluorescently labeled probes bind specifically to their respectivetarget sequences; the 5′ nuclease activity of Taq polymerase cleaves thereporter dye from the probe and a fluorescent signal is generated. Theincrease in fluorescence signal is detected only if the target sequenceis complementary to the probe and is amplified during PCR. A mismatchbetween probe and target greatly reduces the efficiency of probehybridization and cleavage. The ABI Prism 7700HT or 7900HT Sequencedetection System measures the increase in fluorescence during PCRthermal cycling, providing “real time” detection of PCR productaccumulation. Real Time detection on the ABI Prism 7900HT or 7900HTSequence Detector monitors fluorescence and calculates Rn during eachPCR cycle. The threshold cycle, or Ct value, is the cycle at whichfluorescence intersects the threshold value. The threshold value isdetermined by the sequence detection system software or manually.

“RT-PCR” as used herein refers to the combination of reversetranscription and PCR in a single assay. “Reverse transcription” is aprocess whereby an RNA template is transcribed into a DNA molecule by areverse transcriptase enzyme. Thus, “reverse transcriptase” describes aclass of polymerases characterized as RNA-dependent DNA polymerases,that is, such polymerases use an RNA template to synthesize a DNAmolecule. Historically, reverse transcriptases have been used toreverse-transcribe mRNA into cDNA. However, reverse transcriptases canbe used to reverse-transcribe other types of RNAs such as viral genomicRNA or viral sub-genomic RNA. Standard reverse transcriptases includeMaloney Murine Leukemia Virus Reverse Transcriptase (MoMuLV RT) andAvian myoblastosis virus (AMV). These enzymes have 5′->3′ RNA-dependentDNA polymerase activity, 5′->3′ DNA-dependent DNA polymerase activity,and RNase H activity. However, unlike many DNA-dependent DNApolymerases, these enzymes lack 3′->5′ exonuclease activity necessaryfor “proofreading,” (i.e., correcting errors made during transcription).After a DNA copy of an RNA has been prepared, the DNA copy may besubjected to various DNA amplification methods such as PCR.

LCR is a method of DNA amplification similar to PCR, except that it usesfour primers instead of two and uses the enzyme ligase to ligate or jointwo segments of DNA. LCR can be performed as according to Moore et al.,Journal of Clinical Microbiology 36(4)1028-1031 (1998). Briefly, an LCRreaction mixture contains two pair of primers, dNTP, DNA ligase and DNApolymerase representing about 90 μl, to which is added 100 μl ofisolated nucleic acid from the target organism. Amplification isperformed in a thermal cycler (e.g., LCx of Abbott Labs, North Chicago,Ill.).

TAS is a system of nucleic acid amplification in which each cycle iscomprised of a cDNA synthesis step and an RNA transcription step. In thecDNA synthesis step, a sequence recognized by a DNA-dependent RNApolymerase (i.e., a polymerase-binding sequence or PBS) is inserted intothe cDNA copy downstream of the target or marker sequence to beamplified using a two-domain oligonucleotide primer. In the second step,an RNA polymerase is used to synthesize multiple copies of RNA from thecDNA template. Amplification using TAS requires only a few cyclesbecause DNA-dependent RNA transcription can result in 10-1000 copies foreach copy of cDNA template. TAS can be performed according to Kwoh etal., PNAS 86:1173-7 (1989). Briefly, extracted RNA is combined with TASamplification buffer and bovine serum albumin, dNTPs, NTPs, and twooligonucleotide primers, one of which contains a PBS. The sample isheated to denature the RNA template and cooled to the primer annealingtemperature. Reverse transcriptase (RT) is added the sample incubated atthe appropriate temperature to allow cDNA elongation. Subsequently T7RNA polymerase is added and the sample is incubated at 37° C. forapproximately 25 minutes for the synthesis of RNA. The above steps arethen repeated. Alternatively, after the initial cDNA synthesis, both RTand RNA polymerase are added following a 1 minute 100° C. denaturationfollowed by an RNA elongation of approximately 30 minutes at 37° C. TAScan be also be performed on solid phase as according to Wylie et al.,Journal of Clinical Microbiology, 36(12):3488-3491 (1998). In thismethod, nucleic acid targets are captured with magnetic beads containingspecific capture primers. The beads with captured targets are washed andpelleted before adding amplification reagents which containsamplification primers, dNTP, NTP, 2500 U of reverse transcriptase and2500 U of T7 RNA polymerase. A 100 μl TMA reaction mixture is placed ina tube, 200 μl oil reagent is added and amplification is accomplished byincubation at 42° C. in a waterbath for one hour.

NASBA is a transcription-based amplification method which amplifies RNAfrom either an RNA or DNA target. NASBA is a method used for thecontinuous amplification of nucleic acids in a single mixture at onetemperature. For example, for RNA amplification, avian mycloblastosisvirus (AMV) reverse transcriptase, RNase H and T7 RNA polymerase areused. This method can be performed as according to Heim, et al., NucleicAcids Res., 26(9):2250-2251 (1998). Briefly, an NASBA reaction mixturecontains two specific primers, dNTP, NTP, 6.4 U of AMV reversetranscriptase, 0.08 U of Escherichia coli Rnase H, and 32 U of T7 RNApolymerase. The amplification is carried out for 120 min at 41° C. in atotal volume of 201.

In a related method, self-sustained sequence-replication (3SR) reaction,isothermal amplification of target DNA or RNA sequences in vitro usingthree enzymatic activities: reverse transcriptase, DNA-dependent RNApolymerase and Escherichia coli ribonuclease H. This method may bemodified from a 3-enzyme system to a 2-enzyme system by using humanimmunodeficiency virus (HIV)-1 reverse transcriptase instead of avianmyeloblastosis virus (AMV) reverse transcriptase to allow amplificationwith T7 RNA polymerase but without E. coli ribonuclease H. In the2-enzyme 3SR, the amplified RNA is obtained in a purer form comparedwith the 3-enzyme 3SR (Gebinoga & Oehlenschlager European Journal ofBiochemistry, 235:256-261, 1996).

SDA is an isothermal nucleic acid amplification method. A primercontaining a restriction site is annealed to the template. Amplificationprimers are then annealed to 5′ adjacent sequences (forming a nick) andamplification is started at a fixed temperature. Newly synthesized DNAstrands are nicked by a restriction enzyme and the polymeraseamplification begins again, displacing the newly synthesized strands.SDA can be performed as according to Walker, et al., PNAS, 89:392-6(1992). Briefly, an SDA reaction mixture contains four SDA primers,dGTP, dCTP, TTP, dATP, 150 U of Hinc II, and 5 U ofexonuclease-deficient of the large fragment of E. coli DNA polymerase I(exo.sup.-Klenow polymerase). The sample mixture is heated 95° C. for 4minutes to denature target DNA prior to addition of the enzymes. Afteraddition of the two enzymes, amplification is carried out for 120 min.at 37° C. in a total volume of 50 μl. Then, the reaction is terminatedby heating for 2 minutes at 95° C.

Boomerang DNA amplification (BDA) is a method in which the polymerasebegins extension from a single primer-binding site and then makes a looparound to the other strand, eventually returning to the original primingsite on the DNA. BDA is differs from PCR through its use of a singleprimer. This method involves an endonuclease digestion of a sample DNA,producing discrete DNA fragments with sticky ends, ligating thefragments to “adapter” polynucleotides (comprised of a ligatable end andfirst and second self-complementary sequences separated by a spacersequence) thereby forming ligated duplexes. The ligated duplexes aredenatured to form templates to which an oligonucleotide primer annealsat a specific sequence within the target or marker sequence of interest.The primer is extended with a DNA polymerase to form duplex productsfollowed by denaturation of the duplex products. Subsequent multiplecycles of annealing, extending, and denaturing are performed to achievethe desired degree of amplification (U.S. Pat. No. 5,470,724).

The Q-beta replication system uses RNA as a template. Q-beta replicasesynthesizes the single-stranded RNA genome of the coliphage Qβ. Cleavingthe RNA and ligating in a nucleic acid of interest allows thereplication of that sequence when the RNA is replicated by Q-betareplicase (Kramer & Lizardi Trends Biotechnol. 1991 9(2):53-8, 1991).

A variety of amplification enzymes are well known in the art andinclude, for example, DNA polymerase, RNA polymerase, reversetranscriptase, Q-beta replicase, thermostable DNA and RNA polymerases.Because these and other amplification reactions are catalyzed byenzymes, in a single step assay that the nucleic acid releasing reagentsand the detection reagents should not be potential inhibitors ofamplification enzymes if the ultimate detection is to be amplificationbased.

Amplification of the nucleic acid molecules in a nucleic acid sample canresult replication of at least 0.01% of the nucleic acid sequences inthe nucleic acid sample, at least 0.1% of the nucleic acid sequences inthe nucleic acid sample, at least 1% of the nucleic acid sequences inthe nucleic acid sample, at least 5% of the nucleic acid sequences inthe nucleic acid sample, at least 10% of the nucleic acid sequences inthe nucleic acid sample, at least 20% of the nucleic acid sequences inthe nucleic acid sample, at least 30% of the nucleic acid sequences inthe nucleic acid sample, at least 40% of the nucleic acid sequences inthe nucleic acid sample, at least 50% of the nucleic acid sequences inthe nucleic acid sample, at least 60% of the nucleic acid sequences inthe nucleic acid sample, at least 70% of the nucleic acid sequences inthe nucleic acid sample, at least 80% of the nucleic acid sequences inthe nucleic acid sample, at least 90% of the nucleic acid sequences inthe nucleic acid sample, at least 95% of the nucleic acid sequences inthe nucleic acid sample, at least 96% of the nucleic acid sequences inthe nucleic acid sample, at least 97% of the nucleic acid sequences inthe nucleic acid sample, at least 98% of the nucleic acid sequences inthe nucleic acid sample, or at least 99% of the nucleic acid sequencesin the nucleic acid sample.

The various sequence representations described above and elsewhereherein can be, for example, for 1 target sequence, 2 target sequences, 3target sequences, 4 target sequences, 5 target sequences, 6 targetsequences, 7 target sequences, 8 target sequences, 9 target sequences,10 target sequences, 11 target sequences, 12 target sequences, 13 targetsequences, 14 target sequences, 15 target sequences, 16 targetsequences, 17 target sequences, 18 target sequences, 19 targetsequences, 20 target sequences, 25 target sequences, 30 targetsequences, 40 target sequences, 50 target sequences, 75 targetsequences, or 100 target sequences. The sequence representation can be,for example, for at least 1 target sequence, at least 2 targetsequences, at least 3 target sequences, at least 4 target sequences, atleast 5 target sequences, at least 6 target sequences, at least 7 targetsequences, at least 8 target sequences, at least 9 target sequences, atleast 10 target sequences, at least 11 target sequences, at least 12target sequences, at least 13 target sequences, at least 14 targetsequences, at least 15 target sequences, at least 16 target sequences,at least 17 target sequences, at least 18 target sequences, at least 19target sequences, at least 20 target sequences, at least 25 targetsequences, at least 30 target sequences, at least 40 target sequences,at least 50 target sequences, at least 75 target sequences, or at least100 target sequences.

The sequence representation can be, for example, for 1 target sequence,2 different target sequences, 3 different target sequences, 4 differenttarget sequences, 5 different target sequences, 6 different targetsequences, 7 different target sequences, 8 different target sequences, 9different target sequences, 10 different target sequences, 11 differenttarget sequences, 12 different target sequences, 13 different targetsequences, 14 different target sequences, 15 different target sequences,16 different target sequences, 17 different target sequences, 18different target sequences, 19 different target sequences, 20 differenttarget sequences, 25 different target sequences, 30 different targetsequences, 40 different target sequences, 50 different target sequences,75 different target sequences, or 100 different target sequences. Thesequence representation can be, for example, for at least 1 targetsequence, at least 2 different target sequences, at least 3 differenttarget sequences, at least 4 different target sequences, at least 5different target sequences, at least 6 different target sequences, atleast 7 different target sequences, at least 8 different targetsequences, at least 9 different target sequences, at least 10 differenttarget sequences, at least 11 different target sequences, at least 12different target sequences, at least 13 different target sequences, atleast 14 different target sequences, at least 15 different targetsequences, at least 16 different target sequences, at least 17 differenttarget sequences, at least 18 different target sequences, at least 19different target sequences, at least 20 different target sequences, atleast 25 different target sequences, at least 30 different targetsequences, at least 40 different target sequences, at least 50 differenttarget sequences, at least 75 different target sequences, or at least100 different target sequences.

Detection

Products of amplification can be detected using any nucleic aciddetection technique. For real-time detection, the amplification productsand the progress of amplification are detected during amplification.Real-time detection is usefully accomplished using one or more or one ora combination of fluorescent change probes and fluorescent changeprimers. Other detection techniques can be used, either alone or incombination with real-timer detection and/or detection involvingfluorescent change probes and primers. Many techniques are known fordetecting nucleic acids. The nucleotide sequence of the amplifiedsequences also can be determined using any suitable technique.

For example, nucleic acid product may be detected by any of a variety ofwell-known methods, for example, electrophoresis (e.g., gelelectrophoresis or capillary electrophoresis). Amplified fragments maybe subjected to further methods of detecting, for example, variantsequences (e.g., single nucleotide polymorphisms (SNPs)). An exemplarymethod is single nucleotide primer extension (Lindblad-Toh et al.,Large-scale discovery and genotyping of single-nucleotide polymorphismsin the mouse. Nature Genet. 2000 April; 24(4):381-6). In this reaction,an oligonucleotide primer is designed to have a 3′ end that is onenucleotide 5′ to a specific mutation site. In some embodiments, theextension primers are labeled with a tag or a member of a binding pairto allow the capture of the primer on solid phase. In particularembodiments, the primers may be tagged with varying lengths ofnonspecific polynucleotides (e.g., poly-GACT) to allow multiplexdetection of preferably 2 or more, more preferably 3 or more, 4 or more,5 or more, even 10 or more different mutations (polymorphisms) in asingle reaction. The primer hybridizes to the PCR amplicon in thepresence of one or more labeled ddNTPs and a DNA polymerase. Thepolymerase extends the primer by one nucleotide, adding a single,labeled ddNTP to the 3′ end of the extension primer. The addition of adideoxy nucleotide terminates chain elongation. If more than onedideoxynucleotide (e.g., ddATP, ddGTP, ddCTP, ddTTP, ddUTP, etc.) isused in a reaction, one or more can be labeled. If multiple labels areused, the labels can be distinguishable e.g., each is labeled with adifferent fluorescent colored dye. The products are labeledoligonucleotides, each one of which may be detected based on its label.Further methods of detecting variant sequences include the READIT SNPGenotyping System (Promega Corporation, Madison Wis.) andoligonucleotide ligation assays.

Examples Example I: Primers for Malaria

Capture sequences were designed with a Tm of 2 to 5° C. below thereaction temperature of 55° C. Primer sequences were designed with a Tmof around 10° C. below the reaction temperature. Linkers attaching the5′ end of the primer to the 5′ end of the probe were used (see FIG. 5).

(SEQ ID NO: 1) 3′ TCGCTACGCA 5′ [Spacer 18][Spacer 18] [Spacer 18] 5′[T(FAM)] (SEQ ID NO: 2) ACGGTGAACTCTCA 3′ [DABCYL] 3′ (SEQ ID NO: 3) 3′TCGCTACGCA 5′ [Spacer 18][Spacer 18] [Spacer 18][Spacer 18] 5′(SEQ ID NO: 4) ACGGTGAACTCTCA 3′ [DABCYL] 3′ (SEQ ID NO: 5) 3′TCGCTACGCA 5′ [Spacer 18][Spacer 18] [Spacer 18][Spacer 18] 5′(SEQ ID NO: 6) TCTAACGGTGAACTC 3′ [DABCYL] 3′

A regular primer was used for the reverse primer and a control usingjust a regular primer for the forward primer and a Rapid Probe fordetection was used. The primers were run in a real-time PCR reactionwith GoTaq DNA master mix with final MgCl2 concentration of 5 mM. Finalprimer concentration was 250 nM. Reaction conditions were 95° C. for 20s followed by 45 cycles of 95° C. for is and 55° C. for 20 s.

All three cooperative primers generated detectable amplicon and had adetectable signal from the labeled capture sequence. The cooperativeprimer with no distance between the primer and capture sequenceamplified less efficiently than the others.

The same real-time PCR reaction was repeated with an annealing/extensiontemperature of 50° C. The signal generated from the labeled capturesequence was greater for all three cooperative primers. Real-time PCRefficiency did not appear to improve at the lower temperature.

Example 2: High Tm Capture Sequences

Labeled capture sequences were designed with a Tm of 7 to 10° C. abovethe reaction temperature of 55° C. The reverse cooperative primer wasmade with an unlabeled capture sequence with a Tm of about 2° C. lessthan the reaction temperature. Primer sequences were designed with a Tmof around 7 to 10° C. below the reaction temperature.

(SEQ ID NO: 7) 3′ TCGCTACGCA 5′ [Spacer 18][Spacer 18] [Spacer 18] 5′[T(FAM)] (SEQ ID NO: 8) ACGGTGAACTCTCATTCCA 3′ [DABCYL] 3′(SEQ ID NO: 9) 3′ TCGCTACGCA 5′ [Spacer 18][Spacer 18] [Spacer 18] 5′[(FAM)] (SEQ ID NO: 10) ACGGTGAACTCTCATTCCA CCG 3′ [DABCYL] 3′(SEQ ID NO: 11) 3′ ATTGACATACCTGC 5′ [Spacer 18][Spacer 18] [Spacer 18]5′ (SEQ ID NO: 12) AGCAAGTGGAATGTT [Phos] 3′

The primers were run in a real-time PCR reaction with GoTaq DNA mastermix with final MgCl2 concentration of 5 mM. Final primer concentrationwas 250 nM. Reaction conditions were 95° C. for 20 s followed by 50cycles of 95° C. for is and 55° C. for 20 s. The real-time PCR was alsorepeated with an extension step of 40 s.

The cooperative primers with High Tm capture sequences had a similaramplification efficiency and change in fluorescence to the low Tmcapture sequences from Example 1. Increasing the extension time did notappear to increase amplification efficiency.

Example 3: Elimination of Primer-Dimers

Primer-dimers were synthesized for the cooperative primers and thenormal primers. Either 0, 600, 6,000 or 600,000 primer-dimers werespiked into a reaction containing 60 copies of Malaria DNA. The primerswere run in a real-time PCR reaction with GoTaq DNA master mix withfinal MgCl2 concentration of 5 mM. Final primer concentration was 250nM. Reaction conditions were 95° C. for 20 s followed by 50 cycles of95° C. for is and 55° C. for 20 s.

The control with normal primers had easily visible positives when noprimer-dimers were spiked in. However, with as few as 600 primer-dimersspiked in, the signal disappeared resulting in false negatives. Incontrast, the cooperative primers had no signal dampening or loss ofamplification product with even as many as 600,000 primer-dimers spikedin.

When a 2.2% Lonza flashgel was run with the PCR products, the gelconfirmed the fact that no primer-dimers were amplified for thecooperative primers. However, the normal primers clearly amplified theprimer-dimers rather than the Malaria DNA, resulting in false negatives.

Example 4: Cooperative Primers with Detection Mechanism on the Primer

Cooperative Primers with a detection mechanism on the primer were made:

(SEQ ID NO: 13) 3′ [Spacer 3] TTGTAAGGTGAACGA 5′ (SEQ ID NO: 46) 5′[Spacer 18][T(FAM)] actgtatgg (SEQ ID NO: 14) [T(BHQ-1)][Spacer 9]CGTCCATACAGTTA 3′ (SEQ ID NO: 15) 3′ [Spacer 3] TTGTAAGGTGAACGA 5′(SEQ ID NO: 47) 5′ [Spacer 9][Spacer 18][T(FAM)] atggacg (SEQ ID NO: 16)[T(BHQ-1)][Spacer 9)] CGTCCATACAGTTA 3′ (SEQ ID NO: 17) 3′ [Spacer 3]TTGTAAGGTGAACGA 5′ (SEQ ID NO: 48) 5′ [T(FAM][Spacer 3] taactgtatg(SEQ ID NO: 18) [T(FAM)][Spacer 18] CGTCCATACAGTTA 3′ (SEQ ID NO: 19) 3′[Spacer 3] TTGTAAGGTGAACGA 5′ (SEQ ID NO: 49) [Spacer 9][T(FAM)]actgtatgg (SEQ ID NO: 20) [T)BHQ-10)[Spacer 18] CGTCCATACAGTTA 3′(SEQ ID NO: 21) 3′ [Spacer 3] AGATTGTAAGGTGAACGA 5′ (SEQ ID NO: 65) 5′[Spacer 18][T(FAM)] actgtatgg (SEQ ID NO: 22) [T(BHQ-1)][Spacer 9]CGTCCATACAGTTA 3′ (SEQ ID NO: 23) 3′ [Spacer 3] TTGTAAGGTGAACGA 5′(SEQ ID NO: 66) 5′ [Spacer 18][T(FAM)] actgtatgg (SEQ ID NO: 24)[T(BHQ-1)][Spacer 9 ]CGTCCATACAGTTAT 3′

Example 5: Labeling the Capture Sequence

P. falciparum real-time PCR was run by making a master mix with 250 nMfinal concentration of each primer (either PfcF inv, PfcF inv62, PfcFinv62HP or PfcF with PfcR), 5 mM final concentration of MgCl2 and anadditional 0.25 U/reaction of GoTaq polymerase (Promega) in GoTaqColorless Master Mix (Promega). 5,000,000, 600,000, 50,000, 500 or 0copies of template were added to each reaction. The reaction was run onthe ABI StepOne and included a 20 s denature step at 95° C. followed by45 cycles of 95° C. for 1 s and 55° C. for 20 s. Each reaction was runin duplicate.

Having demonstrated that cooperative primers are capable of efficientamplification and can eliminate interference from primer-dimers, weattempted to incorporate a probe into the primer. This was done bylabeling the capture sequence. First, inverted primers were attached tothe 5′ end of capture sequences having Tm's both below and above thereaction temperature, including capture sequences with hairpin formationto encourage greater quenching of the fluorophore (Pf cF inv, Pf cF inv62 and Pf cF inv 62HP). However, very little signal was observed fromthese primers and electrophoretic gels showed that very few of theprimers were cleaving the capture sequence (FIG. 8—the barely visiblebands below the amplicon of the cooperative primers).

It was believed that conformational stain from the linker was liftingthe 5′ end of the capture sequence and causing the polymerase todisplace the sequence rather than cleave it. Consequently, if the strainwas moved from the 5′ end to the 3′ end (e.g. by changing where thelinker was attached), the polymerase might cleave the capture sequencewith greater efficiency. Upon testing this hypothesis the fluorescentsignal rose dramatically (FIG. 8). Even though the labeled capturesequence had a Tm below the reaction temperature, the signal was still2.5 times higher than the signal from normal hybridization probes.

Example 6: SNP Differentiation

M. tuberculosis real-time PCR for the D516V mutation in the rpoB geneconferring rifampicin resistance was run by making a master mix with 250nM final concentration of each primer/probe (MTb cF, MTb P, and one ofMTb cR1, MTb cR2, MTb cR3, MTb cR4, MTb cR5, MTb cR6, MTb cR7, MTb cR8or MTb cR9), 5 mM final concentration of MgCl2 and an additional 0.25U/reaction of GoTaq polymerase (Promega) in GoTaq Colorless Master Mix(Promega). 50,000 copies of template (MTb WT or MTb D516V) were added toeach reaction. Each reaction was run in duplicate. The reaction was runon the ABI 7500 and included a 20 s denature step at 95° C. followed by45 cycles of 95° C. for 3 s and 55° C. for 3 s. The Ct's wereautomatically determined by the machine with a threshold of 10,000 andthe ΔRn was taken from cycle 45 of the exported data.

Finally, the ability of these efficient, primer-dimer free, cooperativeprimers to differentiate SNP's was analyzed. Cooperative Primers weredesigned to the rpoB gene D516V mutation, which is present in up to 7.4%of rifampicin resistant M. tuberculosis isolates in India. Two differentstrategies were employed: 1) the ARMS method and 2) labeled capturesequence differentiation. Both methods resulted in the ability todifferentiate SNP's similar to standard primers and probes (FIG. 9 anddata summary in Table 1).

For the probe based method, cooperative primer MTb cR6 gave the bestratio of fluorescent signals between the mutant and wild type strains.For the ARMS based method, MTb cR8 gave the best difference in Ctvalues. Both are shown in FIG. 9.

TABLE 1 D516V primer Primer Probe ΔRnVar/ name Method ΔTm ΔTm ΔCt ΔRnWTMTb cR1 Probe (4.1) 4.5 1.68 3.01 MTb cR2 Probe (4.1) (2.5) 4.79 3.35MTb cR5 Probe (6.6) (2.5) 4.83 1.89 MTb cR6 Probe (4.1) (7.1) 3.83 3.67MTb cR7 Probe (4.1) (11.7) 5.30 3.62 MTb cR3 ARMS (6.3) 4.3 4.43 n/a MTbcR4 ARMS (10.2) 4.3 5.99 n/a MTb cR8 ARMS (20.2) 4.3 7.57 n/a MTb cR9ARMS (25.5) 4.3 7.13 n/a

Table 1 Shows a Summary of SNP Differentiation Methods.

Each primer is listed together with whether it uses ARMS or probe(labeled capture sequence) based differentiation, the number of degreesthe predicted Tm for the primer or probe is above or below the reactiontemperature (values below the reaction temperature are in red font andin parenthesis), the difference between mutant and wild type Ct values,and the ratio of the mutant and wild type fluorescence.

Sequences 5′ to 3′ Beta Actin (Amplification Efficiency)Normal Primers/Probes b-act P [FAM] TGTGGCCGAGGACTTTGAcggc [BHQ1](SEQ ID NO: 25) Cooperative Primers 3′ AGTGGCAAGGTC 5′(SEQ ID NO: 26) [Sp18][Sp18][Sp18] b-act cF 5′ GGTGACAGCAGTC [Sp3]3′(SEQ ID NO: 27) 3′ TAGGATTTTCGGTG 5′(SEQ ID NO: 28) [Sp18][Sp18][Sp18] b-act cR 5′ GCAAGGGACTTCC [Sp3] 3′(SEQ ID NO: 29) Templates Beta actinAGGATTTAAAAACTGGAACGGTGAAGGTGACAGCAGTCGGTTGGAGCGAGCATCCCCCAAAGTTCACAATGTGGCCGAGGACTTTGATTGCACATTGTTGTTTTTTTAATAGTCATTCCAAATATGAGATGCGTTGTTACAGGAAGTCCCTTGCCATCCTAAAAGCCACCCCA (SEQ ID NO: 30)P. Falciparum (Impact of Primer-Dimers and Probe Selection)Normal Primers/Probes Pf nF CGCATCGCTTCTAACGGTGA (SEQ ID NO: 31) Pf nRGAAGCAAACACTAGCGGTGGAA (SEQ ID NO: 32) Pf P [FAM]ACTCTCATTCCAATGGAACCTTGTTCAAGTTCAAAccattggaa [DABC] (SEQ ID NO: 33)Cooperative Primers/Probes Pf cF inv 3′ TCGCTACGCA (SEQ ID NO: 34) 5′[Sp18][Sp18][Sp18] 5′ [FAM] ACGGTGAACTCTCA [DABC] 3′ (SEQ ID NO: 35)Pf cF inv 3′ TCGCTACGCA 5′ (SEQ ID NO: 36) [Sp18][Sp18][Sp18] 5′[T(FAM)] A CGGTGAACTCTCATTCCA 3′ (SEQ ID NO: 37) 62 Pf cF inv 3′TCGCTACGCA 5′ (SEQ ID NO: 38) [Sp18][Sp18][Sp18] 5′ [T(FAM)]ACGGTGAACTCTCATTCCA ccg [DABC] 3′ (SEQ ID NO: 39) 62HP Pf cF [FAM]ACGGTGAACTCTCA [Sp18][Sp18][Sp18][Sp18][Sp18][Sp18]ACGCATCGCT (SEQ ID NO: 40) Pf cR inv 3′ ATTGACATACCTGC 5′(SEQ ID NO: 67) [Sp18][Sp18][Sp18] 5′ AGCAAGT GGAATGTT [Phos] 3′(SEQ ID NO: 41) Low Tm Primers minus Capture Sequence Pf Low TmF ACGCATCGCT (SEQ ID NO: 42) Pf Low Tm R CGTCCATACAGTTA (SEQ ID NO: 43)Templates Normal GAAGCAAACACTAGCGGTGGAATCACCGTTAGAAGCGATGCG Primer-Dimer(SEQ ID NO: 44) Cooperative CGTCCATACAGTTA AGCGATGCGT (SEQ ID NO: 45)Primer-Dimer P. CCAGCTCACGCATCGCTTCTAACGGTGAACTCTCATTCCAATGGAAFalciparum CCTTGTTCAAGTTCAAATAGATTGGTAAGGTATAGTGTTTACTATCAAATGAAACAATGTGTTCCACCGCTAGTGTTTGCTCTAACATTCCACTTGCTTATAACTGTATGGACG (SEQ ID NO: 50)M. Tuberculosis (SNP Differentiation) Normal Primers/Probes MTb P [FAM]CGCCGCGATCAAGGAGTTCgcg [BHQ1] (SEQ ID NO: 51) Cooperative Primers/ProbesMTb cF 3′ ACACTAGCGGAG 5′ (SEQ ID NO: 52) [Sp18][Sp18][Sp18] 5′CGCAGACGTTGAT [Phos] 3′(SEQ ID NO: 53) MTb cR1 [CF 560]TGGaCCATGAATTGGCT [BHQ1] [Sp18][Sp18][Sp18][Sp18] [Sp18][Sp18]CAGCGGGTTGTT (SEQ ID NO: 54) MTb cR2 [CF 560] TGGaCCATGAATTGG [BHQ1][Sp18][Sp18][Sp18][Sp18] [Sp18][Sp18] CAGCGGGTTGTT (SEQ ID NO: 55)MTb cR3 [CF 560]CATGAATTGGCTCAGCTG [BHQ1] [Sp18][Sp18][Sp18][Sp18][Sp18][Sp18] GGGTTGTTCTGGa (SEQ ID NO: 56) MTb cR4[CF 560]CATGAATTGGCTCAGCTG [BHQ1] [Sp18][Sp18][Sp18][Sp18] [Sp18][Sp18]CGGGTTGTTCTaGa (SEQ ID NO: 57) MTb cR5 [CF 560]TGGaCCATGAATTGG [BHQ1][Sp18][Sp18][Sp18][Sp18] [Sp18][Sp18] AGCGGGTTGTT (SEQ ID NO: 58)MTb cR6 [CF 560]TGGaCCATGAATTG [BHQ1] [Sp18][Sp18][Sp18][Sp18][Sp18][Sp18] CAGCGGGTTGTT (SEQ ID NO: 59) MTb cR7[CF 560]TGGaCCATGAATT [BHQ1] [Sp18][Sp18][Sp18] [Sp18][Sp18][Sp18]CAGCGGGTTGTT (SEQ ID NO: 60) MTb cR8 [CF 560] CATGAATTGGCTCAGCTG [BHQ1][Sp18][Sp18][Sp18][Sp18] [Sp18][Sp18] GGGTTGTTCTcGa (SEQ ID NO: 61)MTb cR9 [CF 560]CATGAATTGGCTCAGCTG [BHQ1] [Sp18][Sp18][Sp18][Sp18][Sp18][Sp18] GGGTTcTTCTGGa (SEQ ID NO: 62) Templates MTb WTCGTGGAGGCGATCACACCGCAGACGTTGATCAACATCCGGCCGGTGGTCGCCGCGATCAAGGAGTTCTTCGGCACCAGCCAGCTGAGCCAATTCATGGACCAGAACAACCCGCTGTCGGGGTTGACCCACAAGCGCCGACTGTCGGCGCTGGGGCCCGGCGGTCTGTCACGTGAGCGTGCCGGGCTGGAGGT CCGCGA (SEQ ID NO: 63)MTb D516V CGTGGAGGCGATCACACCGCAGACGTTGATCAACATCCGGCCGGTGGTCGCCGCGATCAAGGAGTTCTTCGGCACCAGCCAGCTGAGCCAATTCATGGTCCAGAACAACCCGCTGTCGGGGTTGACCCACAAGCGCCGACTGTCGGCGCTGGGGCCCGGCGGTCTGTCACGTGAGCGTGCCGGGCTGGAGG TCCGCGA (SEQ ID NO: 64)

It is understood that the disclosed method and compositions are notlimited to the particular methodology, protocols, and reagents describedas these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “aprimer” includes a plurality of such primers, reference to “the primer”is a reference to one or more primers and equivalents thereof known tothose skilled in the art, and so forth.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, also specifically contemplated and considered disclosed isthe range from the one particular value and/or to the other particularvalue unless the context specifically indicates otherwise. Similarly,when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another,specifically contemplated embodiment that should be considered disclosedunless the context specifically indicates otherwise. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint unless the context specifically indicates otherwise. Finally,it should be understood that all of the individual values and sub-rangesof values contained within an explicitly disclosed range are alsospecifically contemplated and should be considered disclosed unless thecontext specifically indicates otherwise. The foregoing appliesregardless of whether in particular cases some or all of theseembodiments are explicitly disclosed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are specifically incorporated by reference. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such disclosure by virtue of prior invention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

We claim:
 1. A cooperative nucleic acid molecule comprising: a. a firstnucleic acid sequence, wherein the first nucleic acid sequence iscomplementary to a first region of a target nucleic acid, and whereinthe first nucleic acid is extendable on the 3′ end; b. a second nucleicacid sequence, wherein the second nucleic acid sequence is complementaryto a second region of the target nucleic acid, such that in the presenceof the target nucleic acid it hybridizes to the target nucleic aciddownstream from the 3′ end of the first nucleic acid sequence; c. alinker connecting said first and second nucleic acid sequences in amanner that allows both the said first and second nucleic acid sequencesto hybridize to the target at the same time.
 2. The cooperative nucleicacid molecule of claim 1 wherein the first nucleic acid molecule willnot hybridize to the target without the second nucleic acid moleculehybridizing to the target.
 3. The cooperative nucleic acid molecule ofclaim 1 wherein the second nucleic acid molecule will not hybridize tothe target without the first nucleic acid molecule hybridizing to thetarget.
 4. The cooperative nucleic acid molecule of claim 1 whereinneither the first nor the second nucleic acid molecule will hybridize tothe target without the other hybridizing to the target.
 5. Thecooperative nucleic acid molecule of claim 1 wherein the effectivemelting temperature (Tm) of the first nucleic acid molecule is increasedby at least 1° C. as compared to the isolated Tm of the first nucleicacid sequence without the second nucleic acid sequence attached to it.6. The cooperative nucleic acid molecule of claim 1 wherein thecooperative nucleic acid molecule comprises a label.
 7. The nucleic acidof claim 6, wherein the second nucleic acid sequence comprises a label.8. A method for synthesizing a nucleic acid, the method comprising: a.contacting a target nucleic acid with b. a cooperative nucleic acidmolecule comprising: i. a first nucleic acid sequence, wherein the firstnucleic acid sequence is complementary to a first region of a targetnucleic acid, and wherein the first nucleic acid is extendable on the 3′end; ii. a second nucleic acid sequence, wherein the second nucleic acidsequence is complementary to a second region of the target nucleic acid,such that it hybridizes to the target nucleic acid downstream from the3′ end of the first nucleic acid sequence; iii. a linker connecting saidfirst and second nucleic acid sequences in a manner that allows both thesaid first and second nucleic acid sequences to hybridize to the targetat the same time; c. and providing conditions appropriate for nucleicacid synthesis, thereby synthesizing a nucleic acid.
 9. The method ofclaim 8 wherein the cooperative nucleic acid molecule comprises a label.10. The method of claim 8, wherein more than one cooperative nucleicacid molecule with different sequences are provided.
 11. A method fordetecting a target nucleic acid, the method comprising: a. contacting asample containing the target nucleic acid with b. a cooperative nucleicacid molecule comprising: i. a first nucleic acid sequence, wherein thefirst nucleic acid sequence is complementary to a first region of atarget nucleic acid, and wherein the first nucleic acid is extendable onthe 3′ end; ii. a second nucleic acid sequence, wherein the secondnucleic acid sequence is complementary to a second region of the targetnucleic acid, such that it hybridizes to the target nucleic aciddownstream from the 3′ end of the first nucleic acid sequence; iii. alinker connecting said first and second nucleic acid sequences in amanner that allows both the said first and second nucleic acid sequencesto hybridize to the target at the same time; c. and detecting the targetanalyte.
 12. The method of claim 11 wherein the label is attached tosaid second nucleic acid sequence.
 13. The method of claim 12 whereinthe change in signal is derived from the change in signal due tonuclease cleavage of the second nucleic acid sequence.
 14. A method foramplifying a target nucleic acid, the method comprising: a) providingthe cooperative nucleic acid molecule of claim 1; b) providing a targetnucleic acid; and c) amplifying the target nucleic acid underappropriate conditions for amplification The method of claim 14, whereinthe first nucleic acid sequence is a primer.
 15. The method of claim 14,wherein the second nucleic acid sequence is a probe.
 16. The method ofclaim 14, wherein more than one cooperative nucleic acid molecule withdifferent sequences are provided.
 17. The method of claim 14, whereinthe first nucleic acid sequence without the second nucleic acid sequenceattached to it is a normal primer.
 18. A method for detecting a nucleicacid, the method comprising: a) providing the cooperative nucleic acidmolecule of claim 1, wherein the cooperative nucleic acid comprises adetectable label; b) providing a target nucleic acid; and c) detectingthe target nucleic acid.
 19. The method of claim 18, wherein thedetectable label is attached to the first nucleic acid sequence.
 20. Themethod of claim 18, wherein the detectable label is attached to thesecond nucleic acid sequence.